Sialyl lewis-x mimetics containing naphthyl backbones

ABSTRACT

Compounds that possess selectin binding activity are described that have a three-dimensionally stable configuration for sialic acid and fucose, or analogs, derivatives, or mimics of these groups, such that sialic acid and fucose or their mimics are separated by a linker that permits binding between those groups and the selecting, such compounds being represented by the following general structural formula I: ##STR1##

This application is a continuation-in-part of Ser. No. 08/289,715, filedAug. 12, 1994, now U.S. Pat. No. 5,658,880, which is acontinuation-in-part of Ser. No. 08/078,949, filed Jun. 16, 1993, nowabandoned. This application is also a continuation-in-part of Ser. No.08/446,185, filed May 19, 1995. All of these applications areincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

This invention relates generally to the field of medicinal chemistry,and specifically to medicaments that are characterized by their capacityto bind to one or more of the three known selectins: E, L, andP-selectins. The medicaments consist of substitutedpseudo-oligosaccharides wherein three chemical moieties are covalentlylinked in the following order: sialic acid, or an analogue, derivative,or mimic thereof, a naphthyl spacer, and fucose or an analogue,derivative, or mimic thereof. Such medicaments have significantapplications for diagnosis or prophylactic or therapeutic treatment ofcertain diseases including cancer, autoimmunity, and inflammation.

BACKGROUND OF THE INVENTION

A large body of data has been accumulated that establishes a family ofreceptors, the selectins (LEC-CAMs) in certain diseases includingcancer, autoimmunity, and in the inflammatory response. The three knownmembers of this family, L-Selectin (LECAM-1, LAM-1, gp9OMEL), E-Selectin(LECAM-2, ELAM-1) and P-Selectin (LECAM-3, GMP-140, PADGEM), eachcontain a domain with homology to the calcium-dependent lectins(C-lectins), an EGF-like domain, and several complement bindingprotein-like domains (Bevilacqua et al., Science (1989) 243:1160-1165;Johnston et al., Cell (1989) 56:1033-1044; Lasky et al., Cell (1989)56:1045-1055; Tedder et al., J. Exp. Med. (1989) 170:123-133, Dasguptaet al., Exp. Opin. Invest. Drugs (1994) 3(7):709). It has been proposedthat the selectins bind to particular ligands and that this accounts fortheir biological activity. Thus, drugs that interfere with or preventbinding of the ligands to the selectins will be useful medicaments fortreating a variety of diseases.

For instance, adhesion of circulating neutrophils to stimulated vascularendothelium is a primary event of the inflammatory response. Recently,Buerke et al. have demonstrated the important role of selectins ininflammatory states such as ischemia-reperfusion injury in cats (Buerke,M. et al., J. Clin. Invest. (1994) 93:1140). Turunen et al. havedemonstrated the role of sLe^(x) and L-selectin in site-specificlymphocyte extravasation in renal transplants during acute rejection(Turunen, J. P. et al., Eur. J. Immunol. (1994) 24:1130). P-selectin hasbeen shown to be centrally involved particularly as related to acutelung injury. Mulligan et al. have reported strong protective effectsusing anti-P-selectin antibody in a rodent lung injury model. (Mulligan,M. S. et al., J. Clin. Invest., (1991) 90:1600, Mulligan, M. S. et al.,Nature (1993) 364:149). A central role of P-selectin in inflammation andthrombosis has been demonstrated by Palabrica et al. (Palabrica, T. etal., Nature (1992) 359:843).

Of the three selecting, E-selectin is particularly interesting becauseof its transient expression on endothelial cells in response to IL-1 orTNF (Bevilacqua et al., Science (1989) 243:1160). The time course ofthis induced expression(2-8 h) suggests a role for this receptor ininitial neutrophil extravasation in response to infection and injury.Indeed, Gundel et al. have shown that antibody to E-selectin blocks theinflux of neutrophils in a primate model of asthma and thus isbeneficial for preventing airway obstruction resulting from theinflammatory response. (Gundel R. H. et al., J. Clin. Invest. (1991)88:1407).

Several different groups have published papers regarding E-selectinligands. Lowe et al., (1990) demonstrated a positive correlation betweenE-selectin dependent adhesion of HL-60 cell variants and transfectedcell lines, with their expression of the sialyl Lewis x (sLe^(x))oligosaccharide, NeuNAc α-2-3-Gal-β1-4(Fuc α-1-3)-GlcNAc. Bytransfecting cells with plasmids containing an α-(1,3/1,4)fucosyltransferase, they were able to convert non-myeloid COS or CHOlines into sLe^(x) -positive cells that bind in an E-selectin dependentmanner. Walz et al., (1990) were able to inhibit the binding of anE-selectin-IgG chimera to HL-60 cells with a monoclonal antibodydirected against sLe^(x) or by glycoproteins with the sLe^(x) structure,but could not demonstrate inhibition with CD65 or CD15 antibodies. Bothgroups concluded that the sLe^(x) structure is the ligand forE-selectin.

Information regarding the DNA sequences encoding endothelialcell-leukocyte adhesion molecules are disclosed in PCT publishedapplication WO90/13300 published Nov. 15, 1990 incorporated herein byreference. The PCT publication cites numerous articles which may berelated to endothelial cell-leukocyte adhesion molecules. The PCTpublication claims methods of identifying E-selectin ligands, as well asmethods of inhibiting adhesion between leukocytes and endothelial cellsusing such ligands and specifically refers to MILAs which are describedas molecules involved in leukocyte adhesion to endothelial cells. Recentpublications regarding selectin ligands describe the use of L-selectinas an indicator of neutrophil activation (Butcher et al., U.S. Pat. No.5,316,913 issued May 31, 1994), and assays for inhibition of leukocyteadhesion (Rosen et al., U.S. Pat. No. 5,318,890 issued Jun. 7, 1994).

As alluded to above, the ligand for E-selectin, sLe^(x), consists of atleast sialic acid, fucose, and N-acetyl lactosamine. Lactosamineconsists of galactose and 2-amino-2-deoxyglucose. Sialic acid and fucoseare bound to the galactose and glucosamine moieties of lactosamine,respectively. Ligands that bind to the other selectins share similarstructural features. Considering the obvious medical importance ofselectin ligands, significant effort has been, and continues to beexpended to identify the critical physical/chemical parametersassociated with selectin ligands that enhance, or that are required fortheir activity (DeFrees, S. A., et al., J. Am. Chem, Soc., (1993)115:7549). In no small part this effort is being driven by the need tohave selectin ligands that are inexpensive to produce (see U.S. Pat. No.5,296,594 issued Mar. 22, 1994; Allanson, N. M. et al., TetrahedronLett., (1993) 34:3945; Musser, J. H. et al., Current PharmaceuticalDesign (1995) 221-232). It is generally thought that it will beprohibitively expensive to commercially produce naturally occurringsLe^(x) by either enzymatic or chemical synthesis because of the numberof sophisticated reactions involved.

The selectin family of adhesion molecules participate in acuteinflammation by initiating neutrophil rolling on activated endothelialcells. This is particularly evident in studies of ischemia reperfusioninjury, where P-selectin appears to be important in neutrophilrecruitment to damaged tissue. The presence of L-selectin and E- orP-selectin ligands on mononuclear cells has implicated thesereceptor-ligand interactions in chronic inflammation. This has beensupported by the finding of chronic expression of E-selectin indermatologic conditions, and P-selectin expression on joint synovialendothelium derived from rheumatoid arthritis patients. L. Lasky Annu.Rev. Biochem. 64:113-39 (1995); "Selectin Family of Adhesion Molecules"by Michael Forrest and James C. Paulson in Physiology andPathophysiology of Leukocyte Adhesion, Ed. by D. Niel Grangier and DeertSchmid-Schonbein, Oxford University Press, New York, N.Y. (1995).

SUMMARY OF THE INVENTION

A first object of the invention is the description of medicaments thatare selectin ligand mimetics that bind to certain selectins wherein themimetics lack the lactose core structure of the natural selectin ligand,sialyl Lewis^(x) (sLe^(x)), and have substituted in its place a naphthylmoiety, relative to sLe^(x). Such invention compounds are represented bythe following general structural formula I: ##STR2## wherein R¹, R², R³,R⁴, R⁵, R⁶, R⁷, and R⁸ are independently selected from the groupconsisting of

(a) --H, Y--B, alkyl of 1 to 4 carbon atoms optionally substituted with1 to 2 lower alkyl groups,

--W((CH₂)_(n) --B)_(t), --W((CH₂)_(m) --(CHR⁹)_(q) --(CH₂)_(m) --A)_(t);

--OH, lower alkoxy, lower aryloxy, lower aralkoxy, lower alkoxyaryl,amino,

--W((CH₂)_(n) --A)_(t), --O--CH₂ --C.tbd.C--B, --N(Ac)--CH₂--C.tbd.C--B, --NH--CH₂ --C.tbd.C--B, --N(CH₂ --C.tbd.C--B)₂, --N(Ac)CH₂Ar--B, --NHCH₂ Ar--B, --N(CH₂ Ar--B)₂, --OCH₂ Ar--B, --(C═O)(CH₂)_(m)--B,

(b) a direct link to A, and a direct link to B; where

Y--B is selected from the group consisting of ##STR3## A is selectedfrom the group consisting of --(C═O)R¹¹, sialic acid, Kemp's acid,quinic acid, --B, --SO₃ M, --OSO₃ M, --SO₂ NH₂, --PO₃ M'₂, --OPO₃ M'₂,--NO₂, saturated or unsaturated carboxylic acids of 1 to 4 carbon atoms,optionally substituted with 1 to 2 hydroxyl groups, and esters, andamides thereof;

W is selected from the group consisting of a direct link, --O--, --N<,--S--, --NH--, and --NAc--;

B is ##STR4## wherein U is selected from the group consisting of -R⁹,--CH₂ OR¹⁰, --CH₂ O-protecting group, --COOR¹¹, --CON(R¹¹)₂, and --COOM;

R⁹ is lower alkyl;

each n is independently selected from the group 0, 1, 2, and 3;

each m is independently selected from the group 0, 1, 2, 3, and 4;

each q is independently selected from the group 0, 1, and 2;

each s is independently selected from the group 1, 2, and 3;

each z is independently selected from the group 1 and 2;

each t is independently selected from the group 1 and 2, with theproviso that when W is --N<, then t is 2, and for all other definitionsof W, t is 1;

R¹⁰ is selected from the group consisting of --H, -R¹¹, --SO₃ M,--(C═O)R¹¹, --SO₂ NH₂, --PO₃ M'₂, -alk--COOR¹³, alk--CON(R¹¹)₂ and--O-carbohydrate;

R¹¹ is independently selected from the group consisting of --H, loweralkyl, cyclic alkyl of 5 to 6 carbon atoms, heterocyclic alkyl of 4 to 5carbon atoms and 1 to 2 heteroatoms, lower aryl and lower aralkyl;

R¹² is selected from the group consisting of --N(R¹¹)₂, and --SR¹¹ ;

R¹³ is selected from the group consiting of R¹¹, and M;

R¹⁴ is selected from the group consisting of --H, and ---OR¹⁰, with theproviso that when z is 2, then together the two R¹⁴ groups may form adouble bond;

R¹⁵ is independently selected from the group consisting of -R¹¹ and--COOH.

M is selected from the group consisting of Na⁺, K⁺, Mg²⁺, and Ca²⁺ ;

M' is selected from the group consisting of --H, --M, and R⁹ ; and

X is selected from the group consisting of --O--, --S--, --C(R¹¹)₂ --,and --N(R¹¹)--; and pharmaceutically acceptable salts thereof with theprovisos that:

(a) when any of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ are --Y--B and W is adirect link, then at least one adjacent position must be --OH or anether moiety;

(b) no more than two of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ may be Y--Bwhen W is a direct link;

(c) no more than three of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ may be adirect link to A;

(d) only one of R¹, R⁴, R⁵, and R⁸ may be a direct link to B;

(e) when one of R¹, R⁴, R⁵, or R⁸ is a direct link to B, then at mosttwo of the other naphthyl substituents can be a direct link to A when Ais not B;

(f) at most three of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ may beindependently selected from the group consisting of --OH and ethermoieties;

(g) at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ is --H;

(h) when any of R¹, R⁴, R⁵, and R⁸ is a direct link to B, then theadjacent position must be --OH or an ether moiety;

(i) at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ is a substituentcontaining a B group, and at least one is a substituent containing an Agroup where A is not B; and

(j) only when A is directly linked to the naphthyl structure may A be--(C═O)R¹¹, and when A is --(C═O)R¹¹, at least one adjacent positionmust be --OH.

A second object of the invention is a description of certain novelmedicaments that incorporate newly discovered physical/chemicalproperties associated with sLe^(x), such that the medicaments have athree-dimensionally stable configuration for the presentation of thefunctional groups of sLe^(x), sialic acid and fucose, that facilitatesbinding between those groups and the selectins.

A third object of the invention is to provide a composition comprisingselectin ligand medicaments bound to a detectable label and/or bound toa pharmaceutically active drug such as an anti-inflammatory drug.

A fourth object of the invention is to provide a pharmaceuticalformulation containing selectin ligand medicaments which is useful intreating certain diseases.

A fifth object of the invention is to provide a description of methodsto treat or diagnose disease.

A sixth object of the invention is to provide compositions and methodsto determine the site of inflammation by administering labeledformulations of the type referred to above.

Another object of the invention is that the ligands can be labeled andthe labeled ligands used in an assay to detect the presence of selectinsin a sample.

These and other objects, advantages and features of the presentinvention will become apparent to those persons skilled in the art uponreading the details of the synthesis, structure, formulation and usageas more fully set forth below.

Definitions

In accordance with the present invention and as used herein, thefollowing terms are defined with the following meanings, unlessexplicitly stated otherwise.

Sialic acids refer to the family of amino sugars containing 9 or morecarbon atoms, N- and O-substituted derivatives of neuraminic acid.

Kemp's acid refers to 1,3,5-trimethyl-1,3,5-cyclohexane-tricarboxylicacid, where each acid is axial. N-acetyl neuraminic acid refers to5-(acetylamino)-3,5-dideoxy-D-glycero-D-galacto-2-nonulosonic acid:##STR5## α form refers to standard nomenclature representing theconfiguration of the anomeric position of an O- or C-glycoside.

β form refers to standard nomenclature representing the configuration ofthe anomeric position of an O- or C-glycoside.

"Amino" refers to --NR₂ where each R is independently selected from --H,lower alkyl, lower aryl, and lower aralkyl.

"Alkyl" refers to saturated hydrocarbons, which may be straight chain,branched, cyclic, or alicyclic. Preferably the alkyl group contains 1 to10 carbon atoms. Most preferred is 1 to 4 carbon atoms.

"Lower alkyl" refers to branched or straight chain alkyl of 1 to 4carbon atoms.

"Alkoxy" refers to --OR, where R is an alkyl group. Lower alkoxy refersto --OR where R is lower alkyl.

"Aryl" refers to aromatic groups which have one ring having a conjugatedpi electron system and includes carbocyclic aryl, and heterocyclic aryl,both of which may be optionally substituted. Lower aryl refers to anaryl containing up to 6 carbon atoms, and may be optionally substituted.

"Carbocyclic aryl" groups are groups wherein the ring atoms are carbonatoms.

"Heterocyclic aryl" groups are groups having from 1 to 2 heteroatoms inthe ring and the remainder of the ring atoms are carbon atoms. Suitableheteroatoms include nitrogen, oxygen, and sulfur. Suitable heterocyclicaryl groups include pyridyl, furanyl, thienyl, pyrrolyl, and the likeall optionally substituted. Heteroaryl is the same as heterocyclic aryl.

"Alicyclic" refers to groups which combine the properties of aliphaticand cyclic alkyl groups. For example, ##STR6## are alicyclic groups.

The term "optionally substituted" aryl groups refers to either nosubstitution or substitution by one to three substituents independentlyselected from lower alkyl, halo, carboxylic acids, esters, --NO₂, andlower perhaloalkyl.

"Aralkyl" refers to an alkyl group substituted with an aryl group, whichmay be optionally substituted. Benzyl is a suitable aralkyl group. Loweraralkyl refers to up to and including 8 carbon atoms, and may beoptionally substituted. The aralkyl group is attached through the alkylportion of the group.

"Alkenyl" refers to unsaturated groups which contain at least onecarbon-carbon double bond and includes straight-chain, branched chain,and cyclic groups. The double bond may be exo to the chain.

"Alkoxyaryl" refers to aryl substituted with alkoxy group.

"Alkynyl" refers to unsaturated groups which contain at least one carbontriple bond and includes straight-chain, branched chain, and cyclicgroups.

"Aryloxy" refers to --O-aryl.

"Aralkoxy" refers to --O-aralkyl.

"Carboxylic acid" refers to --COOH.

"Ester" refers to --COOR where R is lower alkyl, lower aryl, and loweraralkyl;

"Amide" refers to --CONR₂ where each R is independently selected fromhydrogen, lower alkyl, lower aryl, and lower aralkyl. Preferably atleast one R is hydrogen.

"M" refers to a cationic metal selected from Na⁺, K⁺, Mg²⁺, and Ca²⁺.Where M is associated with --COO⁻, it is preferably a plus one cation,and more preferably Na⁺.

"Protecting group" refers to a group protecting one or several inherentfunctional groups. Suitable "protecting groups" will depend on thefunctionality and particular chemistry used to construct the library.Examples of suitable functional protecting groups will be readilyapparent to skilled artisans, and are described, for example, in Greeneand Wutz, Protecting Groups in Organic Synthesis, 2d ed., John Wiley &Sons, NY (1991), which is incorporated herein by reference. Suitable--O-protecting groups can be found in the above book. Preferred suchprotecting groups include acetate and benzyl.

"Carbohydrate" refers to a chemical moiety comprising the generalcomposition (C)_(n) (H₂ O)_(n), including, but not limited to glucose,galactose, fucose, fructose, saccharose, mannose, arabinose, xylose,sorbose, lactose, and derivatives thereof, including but not limited tocompounds which have other elemental compositions, such as aldonicacids, uronic acids, deoxysugars, or which contain additional elementsor moieties, such as amino sugars wherein n is typically 4, 5, 6, 7atoms and wherein the oxygen atom in the carbohydrate can be replaced bya heteroatom such as nitrogen, sulfur, carbon etc. A carbohydrate asused herein is understood to include chemical structures wherein "H" ofany hydroxy group is replaced by any chemically compatible moiety "R",which can be any monomer, oligomer or polymer in the meaning as usedherein. Carbohydrates can be saturated or unsaturated. Carbohydrates maybe charged or uncharged. Suitable charged carbohydrates includegalacturonic acid, glucuronic acid, and sialic acid.

"Carbohydrate unit" is a monomer comprising a monosaccharide.

"Carbon glycoside" is a carbohydrate derivative wherein the anomericposition does not have an oxygen but a carbon substituent.

"Heteroatom glycoside" is a carbohydrate wherein the oxygen at theanomeric position is replaced by an atom other than oxygen, includingcarbon, nitrogen, sulfur, phosphorous and silicon.

"Identifier tag" is any detectable attribute that provides a means toelucidate the structure of an individual oligomer in a labeled syntheticoligomer library. For example, an identifier tag can be used to identifythe resulting products in the synthesis of a labeled synthetic oligomerlibrary.

"Named Reactions" are chemical reactions which are chemical standardreactions known by those of ordinary skill in the art, including but notlimited to the Alper Reaction, Barbier Reaction, Claisen-IrelandReaction, Cope Rearrangement, Delepine Amine synthesis, GewaldHeterocycle Synthesis, Hiyama-Heathcock Stereoselective Allylation,Stork Radical Cyclization, Trost Cyclopentanation, Weidenhagen ImidazoleSynthesis. See, in general, Hassner and Stumer, 1994. See, among otherplaces, "Organic Syntheses Based on Named Reactions and UnnamedReactions", Tetrahedron Organic Chemistry Series, edts. Baldwin andMagnus, Pergamon, Great Britain.

"Cope Reaction" or "Cope" refers to the 3, 3 sigmatropic rearrangementand includes the Claisen-Ireland Reaction and all forms of the Coperearrangement.

"Oligosaccharide" or "polysaccharide" refers to carbohydrates, includingcarbon glycosides, comprising a plurality of monosaccharides.

"Synthetic chemical library" is a collection of random and semi-randomsynthetic molecules wherein each member of such library is produced bychemical or enzymatic synthesis.

A "Synthesis support" is a material having a rigid or semi-rigid surfaceand having functional groups or linkers. A synthesis support may becapable of being derivatized with functional groups or linkers that aresuitable for carrying out synthesis reactions.

Such materials will preferably take the form of small beads, pellets,disks, capillaries, hollow fibers, needles, solid fibers, cellulosebeads, pore-glass beads, silica gels, polystyrene beads optionallycross-linked with polyethylene glycol divinylbenzene, grafted co-polybeads, poly-acrylamide beads, latex beads, dimethylacrylamide beadsoptionally cross-linked with N,N'-bis-acryloyl ethylene diamine, glassparticles coated with a hydrophobic polymer, or other convenient forms.

"Transformation event" or "Reaction" is any event that results in achange of chemical structure of a compound, monomer, an oligomer orpolymer. A "transformation event" or "reaction" may be mediated byphysical, chemical, enzymatic, biological or other means, or acombination of means, including but not limited to, photo, chemical,enzymatic or biologically mediated isomerization or cleavage, photo,chemical, enzymatic or biologically mediated side group or functionalgroup addition, removal or modification, changes in temperature, changesin pressure, and the like. Thus, "transformation event" or "reaction"includes, but is not limited to, events that result in an increase inmolecular weight of a monomer, an oligomer or polymer, such as, forexample, addition of one or a plurality of monomers, addition of solventor gas, or coordination of metal or other inorganic substrates such as,for example, zeolities. A "transformation event" or "reaction" may alsoresult in a decrease in molecular weight of an oligomer or polymer, suchas, for example, de-hydrogenation of an alcohol to an alkene orenzymatic hydrolysis of an ester or amide. "Transformation events" or"reaction" also include events that result in no net change in molecularweight of a monomer, an oligomer or polymer, such as, for example,stereochemistry changes at one or a plurality of a chiral centers,Claissen rearrangement, Ireland rearrangement, or Cope rearrangement andother events as will become apparent to those skilled in the art uponreview of this disclosure.

"Heterocyclic alkyl" refers to a cyclic alkyl group in which one tothree of the ring atoms are a heteroatom and the remaining ring atomsare carbon atoms. Suitable heteroatoms are nitrogen, oxygen, and sulfur.Suitable heterocyclic alkyl groups are morpholine, piperadine, andpiperazine.

"Adjacent position" refers to the next carbon atom on the naphthyl ring.For example, R³ and R¹ are adjacent to R², in the naphthyl structuredepicted below: ##STR7## R¹ and R¹¹ are not considered adjacentpositions.

"Naphthyl structure" refers to the bicyclic aromatic group above.

"Ether moiety" refers to any substituent or group linked through anoxygen. For example, --O(CH₂)_(n) B, --O(CH₂)_(n) A, and --O(CH₂(C═(R¹¹)₂)CH₂ --B, and alkoxy are ether moieties.

"--Ar--" refers to a phenyl, optionally substituted.

"--alk--" refers to an alkyl linking group which is selected from loweralkyl, and cycloalkyl. Suitable "--alk--" groups include --C(CH₃)₂ --,and ##STR8##

"Halo" refers to halogen atoms --F, --Cl, --Br, and --I.

"Cycloalkyl" refers to cyclic alkyl groups and include cyclopropyl,cyclopentyl, cyclohexyl, and cycloheptyl.

The term "pharmaceutically acceptable salt" includes salts of compoundsof formula I derived from the combination of a compound of thisinvention and an organic or inorganic acid or base. The compounds offormula I are useful in both the free acid and salt form.

BRIEF DESCRIPTION OF THE DRAWINGS

This invention may be better understood and its numerous objects,advantages and features will become apparent to those skilled in the artby reference to the accompanying drawings as follows:

FIG. 1 is a longitudinal schematic view showing the interaction betweenwhite blood cells and activated endothelial cells.

FIG. 2 is a longitudinal schematic view showing how compounds of theinvention would be used as pharmaceuticals to block selectin ligandinteractions.

DETAILED DESCRIPTION OF THE INVENTION

Throughout the description of the invention reference is made to certainpublications including scientific articles and patents or patentapplications. It is the intent that each of these publications beincorporated by reference in their entirety when referred to in thespecification.

Before describing the present invention it is to be understood that thisinvention is not limited to the particular compositions, methods orprocesses described as such compositions and methods may, of course,vary.

As used in this specification and the appended claims, the singularforms "a", "an" and "the" include the plural unless the context clearlydictates otherwise. Thus, for example, reference to "a tetheredcompound" includes mixtures of such compounds, reference to "anE-selectin", "a P-selectin", or "an L-selectin" includes reference torespective mixtures of such molecules, reference to "the formulation" or"the method" includes one or more formulations, methods and/or steps ofthe type described herein and/or which will become apparent to thosepersons skilled in the art upon reading this disclosure.

Some standard abbreviations used in connection with the presentinvention include: BSA, bovine serum albumin; DEAE, diethylaminoethyl;DMSO, dimethylsulfoxide; DMF, N,N-dimethylforamide; DCE, dichloroethane;E-selectin or ELAM-1, endothelial/leukocyte adhesion molecule-1; HPTLC,high performance thin layer chromatography; L-selectin or LECAM-1,leukocyte/endothelial cell adhesion molecule-1; MOPS, 3-N-Morpholino)-propanesulfonic acid; NANA, N-acetylneuraminic acid; PVC,polyvinylchloride; TLC, thin layer chromatography; TFA, trifluoro-aceticacid; Tris, tris (hydroxy-methyl) aminomethane.

Development of the Invention

It is worth noting that while the invention compounds were selected fortheir capacity to bind to certain selecting, and that therefore thisproperty contributes to their medical activity, it cannot, however, beexcluded that they are also exerting their favorable medical effects,either in parallel or in tandem, through additional mechanisms ofaction. Thus, the skilled practitioner of this art will appreciate thata key aspect of the subject invention is the description of novelmedicaments, and that Applicants intend not to be bound by a particularmechanism of action that may account for their prophylactic ortherapeutic effects.

E-selectin has a lectin like domain that recognizes the Sialyl Lewis x(sLe^(x)) tetrasaccharide epitope as shown below in Structure III.##STR9##

The ability of sLe^(x) to bind E-selectin is described by Lowe et al.,Cell (1990) 63:475; Phillips et al., Science (1990) 250-1130; Walz etal., Science (1990) 250:1132; and Tyrrell et al., Proc. Natl. Acad. Sci,USA (1991) 88:10372.

It has also been shown (Berg et al., J. Biol. Chem. (1991) 265:14869;Handa et al., Biochem. Biophys. Res. Commun. (1991) 181:1223) that bothE-selectin and P-selectin recognize the isomeric tetrasaccharide sLe^(a)shown below as Structure IV. ##STR10##

L and P-selectin also bind to sLe^(x) containing ligands, although theseselectins have specificity toward a wider variety of natural ligandscontaining sialylated and sulfated Le^(x), and Le^(a) structures as wellas other sulfated or charged carbohydrates (Varki et al. Proc. Nat'lAcad. Sci. USA 91:7390-7397 (1994).

A key step in developing the compounds of the present invention was therealization that both sLe^(x) and sLe^(a) share a structural similarityin their three dimensional arrangements.

Specifically, we observed that sialic acid and fucose, two functionalepitopes in these tetrasaccharides, are juxtaposed in space in a waysuitable for recognition by the selecting. Most importantly, for bothtetrasaccharides we identified 4 to 12 atoms associated with the lactosecore of the tetrasaccharides that functionally separate sialic acid fromfucose. We postulated that replacement of these atoms would lead tocompounds, such as those described and claimed herein, that maintaintheir selectin binding activity. While 4 to 12 is the preferred numberof atoms, most preferred is 6 to 8 atoms as shown in the figure below.The number of atoms refers to the number of atoms between theO-glycoside of sialic acid and the O-glycoside of fucose.

For instance, a close structural examination of sLe^(x) (shown in III)or a modification thereof wherein R=OH (sLe^(x) Glc) indicates that theepitopes i.e., α-Neu5Ac and L-Fucose, are linked through six atoms (Nos.1-6) or eight atoms (Nos. i-viii) as shown in Structure III (a) below##STR11## wherein R is --NHAc or --OH.

Based on this discovery, we deduced that the corresponding epitopes onthe lectin domain of the selectins, are spaced in a similarthree-dimensional configuration such that maintenance of the 6 to 8atoms in the ligand structure would yield active ligands that aremarkedly different in structure from the naturally occurring ligand.

We have also shown that sLe^(x) and sLe^(a) present the fucose andsialic acid functionalities in a special relationship placing them on asingle face with a spacing of 10-12 Å measured between the carbonylcarbon of the carboxylic acid on sialic acid and the C-3 of fucose. Raoet al. J. Bio. Chem. 269(31):19663 (1994).

The compounds of the present invention possess an acid functionalitymimic which is preferably 8-14 Å, and more preferably 9-11 Å from afucose or fucose mimic. This distance is measured from the carbonylcarbon of the acid mimic to the C-3 carbon of fucose or its equivalenton its mimic.

Additionally, we postulated that replacement of the lactose core with apartially or completely rigid core, while still juxtaposing the twofunctional epitopes in space in a way suitable for recognition by theselectins, would lead to compounds, such as those described and claimedherein, that maintain their selectin binding activity. The Naphthylfamily of compounds offer considerable diversity and facility in termsof attachment of suitable groups to satisfy the spacial requirements forselectin ligand binding.

Using these insights, we then designed certain selectin ligands. Thishas been done by attaching sialic acid and L-fucose as such, or analogs,suitable derivatives, or mimics thereof, through six or eight atoms toprovide a series of compounds shown as structural formula I. This seriesof compounds is designed to competitively inhibit selectins from bindingto their natural ligands. These compounds can be combined withpharmaceutically acceptable excipients to provide pharmaceuticalcompositions useful in a wide range of treatments.

Applicants believe that the carboxylic acid portion of sialic acid isimportant for binding. Thus, mimics of sialic acid include moietiescontaining carboxylic acids, esters and amides. It also includes avinylagous acid which can mimic the acid functionality, such the groupshown below: ##STR12## Other sialic acid mimics, also referred to as "A"in the general description, can be in the form of moieties containingsulfates, sulfonates, phosphates, phosphonates, sulfonamides, nitrates,other carboxylic acid equivlanents, and the like. Other acid mimicsinclude B groups, particularly B groups which contain acids andsulfates.

The compounds of the present invention are designed to provide athree-dimensionally stable configuration for functional groups on sialicacid and fucose moieties or their analogues or mimics so as to allow forbinding between those functional groups and receptors on naturalselectins. The compounds are represented by the following generalstructural formula I: ##STR13## wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, andR⁸ are independently selected from the group consisting of

(a) --H, Y--B, alkyl of 1 to 4 carbon atoms optionally substituted with1 to 2 lower alkyl groups,

--W((CH₂)_(n) --B)_(t), --W((CH₂)_(m) --(CHR⁹)_(q) --(CH₂)_(m) --A)_(t);

--OH, lower alkoxy, lower aryloxy, lower aralkoxy, lower alkoxyaryl,amino,

--W((CH₂)_(r) --A)_(t), --O--CH₂ --C.tbd.C--B, --N(Ac)--CH₂--C.tbd.C--B, --NH--CH₂ --C.tbd.C--B, --N(CH₂ --C.tbd.C--B)₂, --N(Ac)CH₂Ar--B, --NHCH₂ Ar--B, --N(CH₂ Ar--B)₂, --OCH₂ Ar--B, --(C═O) (CH₂)_(m)--B,

(b) a direct link to A, and a direct link to B; where

Y--B is selected from the group consisting of ##STR14## A is selectedfrom the group consisting of --(C═O)R¹¹, sialic acid, Kemp's acid,quinic acid, --B, --SO₃ M, --OSO₃ M, --SO₂ NH₂, --PO₃ M'₂, --OPO₃ M'₂,--NO₂, saturated or unsaturated carboxylic acids of 1 to 4 carbon atoms,optionally substituted with 1 to 2 hydroxyl groups, and esters, andamides thereof;

W is selected from the group consisting of a direct link, --O--, --N<,--S--, --NH--, and --NAc--;

B is ##STR15## wherein U is selected from the group consisting of -R⁹,--CH₂ OR¹⁰, --CH₂ O-protecting group, --COOR¹¹, --CON(R¹¹)₂, and --COOM;

R⁹ is lower alkyl;

each n is independently selected from the group 0, 1, 2, and 3;

each m is independently selected from the group 0, 1, 2, 3, and 4;

each q is independently selected from the group 0, 1, and 2;

each s is independently selected from the group 1, 2, and 3;

each z is independently selected from the group 1 and 2;

each t is independently selected from the group 1 and 2, with theproviso that when W is --N<, then t is 2, and for all other definitionsof W, t is 1;

R¹⁰ is selected from the group consisting of --H, -R¹¹, --SO₃ M,--(C═O)R¹¹, --SO₂ NH₂, --PO₃ M'₂, -alk--COOR¹³, -alk--CON(R¹¹)₂ and--O-carbohydrate;

R¹¹ is independently selected from the group consisting of --H, loweralkyl, cyclic alkyl of 5 to 6 carbon atoms, heterocyclic alkyl of 4 to 5carbon atoms and 1 to 2 heteroatoms, lower aryl and lower aralkyl;

R¹² is selected from the group consisting of --N(R¹¹)₂, and --SR¹¹ ;

R¹³ is selected from the group consiting of R¹¹, and M;

R¹⁴ is selected from the group consisting of --H, and --OR¹⁰, with theproviso that when z is 2, then together the two R¹⁴ groups may form adouble bond;

R¹⁵ is independently selected from the group consisting of -R¹¹ and--COOH.

M is selected from the group consisting of Na⁺, K⁺, Mg²⁺, and Ca²⁺ ;

M' is selected from the group consisting of --H, --M, and R⁹ ; and

X is selected from the group consisting of --O--, --S--, --C(R¹¹)₂ --,and --N(R¹¹)--; and pharmaceutically acceptable salts thereof with theprovisos that:

(a) when any of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ are --Y--B and W is adirect link, then at least one adjacent position must be --OH or anether moiety;

(b) no more than two of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ may be Y--Bwhen W is a direct link;

(c) no more than three of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ may be adirect link to A;

(d) only one of R¹, R⁴, R⁵, and R⁸ may be a direct link to B;

(e) when one of R¹, R⁴, R⁵, or R⁸ is a direct link to B, then at mosttwo of the other naphthyl substituents can be a direct link to A when Ais not B;

(f) at most three of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ may beindependently selected from the group consisting of --OH and ethermoieties;

(g) at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ is --H;

(h) when any of R¹, R⁴, R⁵, and R⁸ is a direct link to B, then theadjacent position must --OH or an ether moiety;

(i) at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ is a substituentcontaining a B group, and at least one is a substituent containing an Agroup where A is not B; and

(j) only when A is directly linked to the naphthyl structure may A be--(C═O)R¹¹, and when A is --(C═O)R¹¹, at least one adjacent positionmust be --OH.

The structures that contain the appropriate reactive functions can bereacted with suitably protected hydrophobic carriers like ceramide or aceramide mimic, steroids, diglycerides or phospholipids to form othermedically useful molecules.

The compounds can act as antagonist ligand molecules, i.e. biochemicalblocking agents by binding to selectins and preventing circulatingleukocytes from binding to endothelial cells, thereby preventing aprimary event involved in certain diseases, including cancer, andparticularly metastatic cancers, conditions associated with acuteinflammation, such as reperfusion injury, septic shock, hypovolemic ortraumatic shock, ARDS, and chronic inflammation diseases such asrheumatoid arthritis and asthma. Agonist ligands have the oppositeeffect.

The compounds of structural formula I can be bound to known drugs, forexample anti-inflammatory drugs so as to target the drug-selectin ligandcomplex to a particular site of disease. Additionally, they can beformulated to provide compositions useful in assaying a sample for thepresence of selectins such as E, L and/or P-selectin, or to detect thesite of inflammation in a patient, or to treat acute inflammation (ortreating the inflammatory symptoms of certain diseases) or otherdiseases involving the interaction of selectins on appropriate celltypes.

Preferred R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ groups include ##STR16##--W(CH₂ --CR¹¹ (OR¹¹)CH₂ --B)_(t), --OH, lower alkoxy, lower aryloxy,lower aryloxy, lower alkoxyaryl, --H, a direct link to A where A is notB, and a direct link to B; where W is a direct link or --O--, and t is1.

Most preferred are ##STR17## --W(CH₂ --CR¹¹ (OR¹¹)CH₂ --B)_(t), --OH,--H, and a direct link to A where A is not B.

The preferred examples of A and B are shown respectively in formula Vand VI. Other examples of A include α or β or other analogues orderivatives of sialic acid other than the N-acetyl neuraminic acidresidue shown in formula V, Kemp's acid, quinic acid, R and S forms ofmandelic acid, R and S forms of glyceric acid, R and S forms of lacticacid, propionic and acetic acid, and esters and amides thereof, --SO₃,--PO₃. The synthesis of certain analogues of sialic acid is described inU.S. Pat. No. 5,138,044. ##STR18##

Preferred embodiments of the ligands of the invention are those whereinthe substituent represented by A or B is an N-acetylneuramyl residue andB is fucose.

Preferred forms of B are the α and β forms of L-fucose as shown informula VI. The moiety B also includes substituted forms of thefollowing α and β-fucose structure VI: ##STR19## wherein Me is a methylgroup, R¹⁷, R¹⁸ and R¹⁹ are each independently --OH, --F, --N(R⁹)₂(wherein R⁹ is lower alkyl). Other moieties for B include modifiedfucosides such as corresponding carboxylic analogues of fucose;inositol; substituted inositol; imidazole; substituted imidazole;benzimidazole; substituted benzimidazole; Guanidine; pentaerythritol;substituted pentaerythritol; and substituted butanes of the formula--CH₂ --CHR¹⁷ --CHR¹⁸ --CH₂ R¹⁹ wherein R¹², R¹⁸, and R¹⁹ areindependently --OH, --F or --N(R⁹)₂.

Preferred B groups include those where s is 1 or 2, R¹⁴ is --H or --OH,X is --O--, U is --CH₂ OR¹⁰ or -R⁹, and R¹⁰ is -alk--COOH, --SO₃ M, --H,or -alk--COOM. Most preferred are those where s is 2. Particularlypreferred B groups include fucose, galactose, mannose, and arabinose.

Preferred s numbers are 1 and 2. Most preferred is 2.

Preferred q numbers are 0 and 1.

Preferred m numbers are 0 and 1.

Preferred n numbers are 0 and 3.

Preferred z numbers are 1.

Preferred R¹⁰ groups are --H, SO₃ M, -alk--COOR¹³, and --O-carbohydrate.Most preferred are --H, --SO₃ M, and -alk--COOR¹³.

Preferred R¹¹ groups are --H, lower alkyl, and lower aralkyl. Mostpreferred is --H.

A preferred R¹² group is --N(R¹¹)₂.

Preferred R¹⁴ groups are --H and --OH.

Preferred R¹⁵ groups are --COOH, --H, and --CH₃.

A preferred M cation is Na⁺.

Preferred M' groups are --H, Na⁺, and --CH₃.

A preferred X group is --O--.

Preferred U groups are --CH₂ OR¹⁰ and -R⁹.

Preferred --W(CH₂ (C═C(R¹⁵)₂)--CH₂ --B)_(t), groups are those where W isa direct link or --O--, t is 1, and R¹⁵ is independently --H, --CH₃, and--COOH.

In one preferred aspect, R³ and R⁶ are selected from the groupconsisting of --SO₃ M, --OSO₃ M, --NO₂, and --CO₂ H, and at least one ofR¹, R², or R⁷ is selected from the group OH and --NH₂.

In another preferred aspect, R¹ is a direct link to B, R² and R³ areselected from the group consisting of ##STR20## where W is a direct linkand t is 1, R⁷ is --H or --OH, and R⁶ is selected from the groupconsisting of --H and --(CH₂)_(m) --(CHR⁹)_(q) --(CH₂)_(m) --A, whereeach m and q are independently selected from 0 or 1, and A is asaturated or unsaturated carboxylic acid of 1 to 4 carbon atoms,optionally substituted with 1 to 2 hydroxy groups, and esters and amidesthereof. More preferred is where in the B group s is 1 or 2, R¹⁴ is --Hor --OH, X is --O--, U is --CH₂ OR¹⁰ or -R⁹, and R¹⁰ is -alk--COOH,--SO₃ M, --H, or -alk--COOM. Also preferred is where R¹⁵ is --H.

Preferred compounds include those prepared in the Examples and thosefound in Table 1. ##STR21## Assaying Compounds of Formula I

The compounds of formula I can be tested for their ability to bind to aselectin receptor and/or block the binding site of the receptor andthereby prevent a natural ligand from binding to the selectin receptor.A generalized procedure for testing the compounds of formula I is givenbelow.

An ELISA assay is preferably used that employs recombinant fusionproteins composed of extracellular portions of the human selectinsjoined to human immunoglobulin heavy chain CH₃, CH₂, and hinge regions.See, for example, Walz et al., Science (1990) 250:1132; Aruffo et al.,Cell (1991) 67:35; Aruffo et al., Proc. Natl. Acad. Sci. USA (1992)89:2292. The assay is well known in the art, and generally consists ofthe following three steps: I. 2,3 sLe^(x) glycolipid (25 picomole/well)was transferred into microliter wells as solutions and then evaporatedoff. Excess, which remained unattached, was washed off with water. Thewells were then blocked with 5% BSA at room temperature for an hour andthen washed with PBS containing 1 mM calcium. II. Preparation of"multivalent" receptor of the Selectin-IgG chimera was carried out bycombining the respective chimera (1 μg/mL) with biotin labelled goatF(ab')₂ anti-human IgG (Fc specific) and streptavidin-alkalinephosphatase diluted 1:1000 in 1% BSA-PBS (1 mM calcium) and incubatingat 37° C. for 15 min. This allowed the soluble multivalent receptorcomplex to form. III. Potential inhibitors such as the compounds offormula I were allowed to react with the soluble receptor at 37° C. for45 min. This test assumes that optimal binding, between the solublephase receptor complex and the inhibitor (non natural ligand), wouldhave occurred within this time frame. This solution was then placed inthe microliter wells that were prepared in step I. The plate wasincubated at 37° C. for 45 minutes to allow the soluble receptor to bindto its natural ligand. In the presence of a strong inhibitor only a fewreceptors should be free to bind to the microliter plate coated with thenatural ligand.

The positive control is the signal produced by the soluble receptor whenit is allowed to react with 2,3 sLe^(x) glycolipid in the microliterwells in the absence of any inhibitor. This was considered 100% binding.The signal produced by the receptor that had been previously treatedwith an inhibitor (recorded as O.D.), was divided by the signal producedby the positive control and multiplied by 100 to calculate the %receptor bound to the well in the presence of the inhibitor. Thereciprocal of this is the % inhibition.

It is important to note that invention compounds include those havingsialic acid and fucose separated by 4-12 atoms, or sialic acid oranalogs, derivatives or mimics of sialic acid separated by 4-12 atomsbound to fucose or analogs, derivatives or mimics thereof.

Referring now to FIG. 1, a longitudinal view of a blood vessel 1 isshown. The vessel wall 2 is lined internally with endothelial cells 3.The endothelial cells 3 can be activated causing the cells 3 tosynthesize E or P-selectin which is displayed in FIG. 1 as a triangularsurface receptor 4. Both red blood cells 5 and white blood cells (6A,6B) flow in the vessel 1. The white blood cells 6 display carbohydrateligands 7 which have chemical and physical characteristics which allowthe ligands 7 to bind to the receptors 4. Once the ligand 7 binds to thereceptor 4, the white blood cell 6 is brought through the vessel wall 2as is shown with the white blood cell 6A. The white blood cells 6Bbrought into the surrounding tissue 8 can have positive effects, such asfighting infection, and negative effects, such as inflammation.

An important aspect of the present invention can be described byreferring to FIG. 2. The compounds of formula I are shown as 7A and canadhere to a selectin such as E, L and/or P-selectin by themselves andcan be formulated into pharmaceutical compositions, which whenadministered will effectively block the E, L and/or P-selectin andprevent the adhesion of a ligand 7 connected to a white blood cell 6. Byadministering pharmaceutically effective amounts of the compounds 7A,some, but not all, of the white blood cells will not reach thesurrounding tissue. By slowing the rate at which the white blood cellsreach the surrounding tissue, inflammation can be prevented and/oralleviated.

The selectin family of adhesion molecules participate in acuteinflammation by initiating neutrophil rolling on activated endothelialcells. This is particularly evident in studies of ischemia reperfusioninjury, where P-selectin appears to be important in neutrophilrecruitment to damaged tissue. The presence of L-selectin and E- orP-selectin ligands on mononuclear cells has implicated thesereceptor-ligand interactions in chronic inflammation. This has beensupported by the finding of chronic expression of E-selectin indermatologic conditions, and P-selectin expression on joint synovialendothelium derived from rheumatoid arthritis patients. L. Lasky Annu.Rev. Biochem. 64:113-39 (1995); "Selectin Family of Adhesion Molecules"by Michael Forrest and James C. Paulson in Physiology andPathophysiology of Leukocyte Adhesion, Ed. by D. Niel Grangier and DeertSchmid-Schonbein, Oxford University Press, New York, N.Y. (1995).

The compounds of formula I may also be labeled using standardradioactive, fluorescent, enzymic or other labels for analytical ordiagnostic purposes.

In order for a ligand of the invention to bind to a selectin receptorsuch as E, L and/or P-selectin receptor the ligand need not include theidentical atoms in the identical configuration as per structural formulaI but must have (1) a relatively stable three dimensional conformationas shown in formula I, or (2) a substantially equivalent configurationto that shown in formula I. The equivalency of any other ligand willrelate to its physical three dimensional structure and the electronconfiguration of the molecule, and in particular the charge relatedcharacteristics presented by the groups present on the A and B moietiesshown in formulae V and VI. ##STR22## Assay to Identify Ligand (General)

Candidate ligands can be assayed for their ability to adhere to E, L orP-selectin. The method comprises attaching candidate ligands of formulaI to a substrate surface and then contacting the substrate surfacethereon with recombinant cells, that are genetically engineered toexpress high levels of E, L or P-selectin, for a sufficient time toallow the cells to adhere to the substrate bearing the candidate ligand.Thereafter, centrifugal force or other appropriate methodology isapplied so as to separate away the cells which do not adhere to thesubstrate. Candidate ligands which adhere to E, L or P-selectin,respectively, are determined via the labels on the cells. Such moleculesare isolated, characterized, and their structure specificallyidentified.

Radiolabeled COS cells expressing cell surface E, L and/or P-selectincan be used as probes to screen compounds of the invention. E, L orP-selectin transfected COS cells will adhere to a subset of compounds ofthe invention which can be resolved on TLC plates or adsorbed on PVCmicroliter wells. Adhesion tests are preferably done under physiologicalconditions. Adhesion to these compounds may require calcium, but willnot be inhibited by heparin, chondroitin sulfate, keratin sulfate, oryeast phospho-mannan (PPME). Monosaccharide composition, linkageanalysis, and FAB mass spectrometry of 5 of the purified compounds willindicate that the ligands for E, L or P-selectin share common structuralcharacteristics which generally relate to the moieties A and B and theposition in which they are held.

Identification of Compounds Which Act as E, L and/or P-selectin LigandsUsing Recombinantly Produced Receptor

A complete cDNA for the E, L and/or P-selectin receptor was obtained byPCR starting with total RNA isolated from IL-1 stimulated humanumbilical vein endothelium. The resulting cDNA was inserted into theCDM8 plasmid (see Aruffo et al., Proc. Natl Acad. Sci. USA (1987)84:8573) and the plasmid amplified in E. coli. Plasmid DNA fromindividual colonies was isolated and used to transfect COS cells.Positive plasmids were selected by their ability to generate COS cellsthat support HL-60 cell adhesion. DNA sequencing positively identifiedone of these clones as encoding for E, L and/or P-selectin (Bevilacquaet al., Science, (1989) 243:1160; Polte et al., Nucleic Acids Res (1990)18:1083; Hession et al., Proc. Natl. Acad. Sci. USA (1990) 87:1673).These publications are incorporated herein by reference for theirdisclosure of E-selectin and genetic material coding for its production.The complete nucleotide sequence of the E-selectin cDNA and predictedamino acid sequence of the E-selectin protein are given in the abovecited article by Bevilacqua et al., which DNA and amino acid sequencesare incorporated herein by reference (see also published PCT patentapplication WO90/13300 which was published Nov. 15, 1990, which isincorporated herein by reference).

COS cells, expressing membrane-bound E, L and/or P-selectin, weremetabolically radiolabeled with T₂ PO₄ (tritiated phosphoric acid).These labeled cells can be used as probes in two assay systems to screenfor recognition of the compounds of formula I. More specifically,compounds of formula I may be adsorbed to the bottoms of PVC microliterwells or resolved on TLC plates. In either assay the compounds may beprobed for their ability to support adhesion of E, L and/orP-selectin-transfected COS cells, untransfected COS cells, or COS cellstransfected with a plasmid containing an irrelevant cDNA, underconditions of controlled detachment force (see Swank-Hill et al., Anal.Biochem. (1987) 183:27; and Blackburn et al., J. Biol. Chem. (1986)261:2873 each of which is incorporated herein by reference to disclosethe details of such assaying methodology).

NSAID or non-steroidal, anti-inflammatory drugs such as naproxen oribuprofen which act as anti-inflammatory agents could be administeredbound to the modified ligand and could be administered systemically insmaller amounts than usual while obtaining an equivalent effect or evengreater anti-inflammatory effect at the site of inflammation. Any otherdrugs which might be attached include, but are not limited to,antibiotics, vasodilators and analgesics. Such a drug delivery systemwould reduce any systemic effect normally caused by the drug in that thedrugs could be administered in amounts of one-half to one-tenth thenormal dose and still obtain the same anti-inflammatory result at thesite of inflammation.

UTILITY

The invention compounds have considerable utility for the treatment ofcertain diseases, as set forth herein. However, this is not their onlyutility. Another utility is identification of particular chemicalmoieties that are responsible for, or contribute to ligand binding tothe different selecting. Using the selectin binding assays describedherein it is readily determined which chemical moieties that make up aselectin ligand contribute to selectin binding.

It is believed that the compounds of the present invention can be usedto treat a wide range of diseases, including diseases such as rheumatoidarthritis and ultiple sclerosis. The compositions of the invention houldbe applicable to treat any disease state wherein the immune system turnsagainst the body causing the white cells to accumulate in the tissues tothe extent that they cause tissue damage, swelling, inflammation and/orpain.

The inflammation of rheumatoid arthritis, for example, is created whenlarge numbers of white blood cells quickly enter the joints in the areaof disease and attack the surrounding tissues.

Formulations of the present invention might also be administered toprevent the undesirable after effects of tissue damage resulting fromheart attacks. When a heart attack occurs and the patient has beenrevived, such as by the application of anticoagulants or thrombolytic(e.g., tPA), the endothelial lining where a clot was formed has oftensuffered damage. When the antithrombotic has removed the clot, thedamaged tissue beneath the clot and other damaged tissue in theendothelial lining which has been deprived of oxygen become activated.The activated endothelial cells then synthesize the E-selectin receptorswithin hours of the cells being damaged. The receptors are extended intothe blood vessels where they adhere to glycolipid ligand molecules onthe surface of white blood cells. Large numbers of white blood cells arequickly captured and brought into the tissue surrounding the area ofactivated endothelial cells, resulting in inflammation, swelling, andnecrosis which thereby decreases the likelihood of survival of thepatient.

In addition to treating patients suffering from the trauma resultingfrom heart attack, patients suffering from actual physical trauma couldbe treated with formulations of the invention in order to relieve theamount of inflammation and swelling which normally result after an areaof the body is subjected to severe trauma. Other disease states whichmight be treatable using formulations of the invention include adultrespiratory distress syndrome and various types of arthritis and asthma.After reading the present disclosure, those skilled in the art willrecognize other disease states and/or symptoms which might be treatedand/or mitigated by the administration of formulations of the presentinvention.

Radiolabeled compounds of the invention may be prepared in a sterile,non-pyrogenic medium and injected into the bloodstream of a patient at adose to be determined in the usual way by the physician or radiologist.After a sufficient period for a good balance to have been reachedbetween (i) specificity of binding to activated endothelium compared tonon-specific distribution and (ii) total amount of compound on activatedendothelium, the compound is imaged in a conventional way, according tothe nature of the label used. Use of radiolabelled compounds of theinvention could be used to diagnose disease, such as the site ofinflammation.

The compounds of the invention could also be used as laboratory probesto test for the presence of a selectin receptor such as a receptor of E,L and/or P-selectin in a sample. Such probes are preferably labeled suchas with a radioactive label. There are a number of known labelsincluding radioactive labeled atoms, e.g. radioactive C, O, N, P, or S,fluorescent dyes and enzyme labels which can be attached to compounds ofthe invention using known procedures. Labels as well as methods ofattaching labels to sugar moieties are disclosed in U.S. Pat. No.4,849,513 issued Jul. 18, 1989 to Smith et al. which patent isincorporated herein by reference to disclose labels and methods ofattaching labels.

Method of Synthesis (General)

The compound of formula I can be made using the general and specificsynthesis schemes and examples described below. However, those skilledin the art will recognize variations thereof which are intended to beencompassed by the present invention. In general, the A and B moietiesof formula I are connected and held in a desired three-dimensionalconfiguration. The compounds of the present invention can be prepared ina number of ways. The following schemes show one preferred method ofpreparing the compounds of the present invention. Scheme 1 shows theattachment of a B group to the naphthyl structure. ##STR23##

Scheme 2 shows further elaboration of the naphthyl structure byattachment of another B group through an ether linker. Class Ielectrophiles are alkyl halide C-glycosides. Class II electrophiles areallylic halide C-glycosides Class II electrophiles form allylic ethers."Sugar" refers to C-glycosides below. ##STR24##

Scheme 3 shows the Claisen Ireland rearrangement of the allylic ether toform a trisubstituted naphthyl structure where the second B group,"sugar₂ " is attached through an alkene linker. ##STR25##

Scheme 4 shows several methods of introducing functionality to a sugar.In all three products, the Sugar, has been functionalized so that this Bgroup acts as an acid moiety. ##STR26##

Scheme 5 shows another method of introducing acidic functionality onto asugar. ##STR27##

Schemes 1-5 also apply to compounds where the naphthyl is replaced by aphenyl or flavanoid structure.

The compound shown above can then be reacted with a fluorescent probe, amultivalent compound, a ceramide, cholesterol or other lipid components,or a pharmaceutically active drug such as an anti-inflammatory drug.

Synthesis of Carbon Glycosides

A vast array of methods for carbon-carbon bond formation at the anomericcarbon are known in the art, which also can be applied to the formationof other heteroatom glycosides, as carbon-phosphorous, carbon-sulfur,carbon-nitrogen, or carbon-silicon bonds at the anomeric position whichare understood to be within the invention. The most common method forcarbon-carbon bond formation at the anomeric carbon involvesnucleophilic attack on this electrophilic center. A wide variety ofelectrophilic sugars have been employed, such as reducing sugars (orlactols), alkyl glycosides, anomeric esters, glycosyl halides, imidates,anomeric trichloroacetimidates, glycals, lactones, thioglycosides, aswell as oxygen-protected glycosides such as acetates andp-nitrobenzoates. The carbon nucleophiles that have been used includesilyl enol ethers, alkenes, allylsilanes, allylstannanes, cyanides,homoenolates, and organometallics such as Grignard reagents,organolithiums, cuprates, and aluminates. Further, procedures tosynthesize carbon-glycosides based on metals (palladium, manganese,rhodium, and cobalt) have been developed. Concerted reactions such as4+2! cycloadditions and sigmatropic rearrangements have also beenemployed to generate carbon glycosides. Also, the Wittig Reaction hasextensively been applied to carbon glycoside synthesis, which can bepursued by reaction of hemiacetals followed by ring closure, reaction ofsugar lactones, or reaction of anomeric phosphoranes. Other approachesfor the synthesis of carbon glycosides encompass, among others,palladium mediated reactions, free radical reactions, and reactionsrelying on the electrophilic activity of the anomeric center of sugarmolecules. Special merits of free radical methods are mild reactionconditions and tolerance of a wide range of functional groups. Thesubject of carbon-glycoside synthesis has been reviewed by Hanessian andPernet, 1976, Adv. Chem. Biochem. 33:111; Postema, 1992, Tetrahedron48:8545; Postema, C-Glycoside Synthesis, 1995, CRC Press, Ann Arbor,Mich. Suhadoluid, 1970, Nucleoside Antibiotics Wiley-Interscience: NewYork; Daves and Cheng, 1976, Prog. Med. Chem. 13:303; Inch, 1984,Tetrahedron 40:3161; Hacksell and Daves, 1985, Prog. Med. Chem. 22:1;and Buchanan, 1983, Prog. Chem. Org Natl. Prod. 44:243; and Levy andTang, 1995 "The Chemistry of C-glycosides, Pergamon Press.

The following scheme shows the general chemical reaction underlying thegeneration of activated carbon glycosides useful for the generation ofnovel compounds provided by the present invention: ##STR28##

Reagents effective for the preparation of carbon glycosides includeallyltrimethylsilane (Herscovici and Antonakis, 1992, Nat. Prod. Chem.10:337; Postema, 1992, Tetrahedron 48:8545; Daves, 1990, Acc. Chem. Res.23:201; Hacksell, 1985, Progress in Medicinal Chemistry 22:1; Hanessianand Pernet, 1976, Adv. Chem. Biochem. 33:111; Carbohydrate Chemistry,Specialist Periodical Reports, Royal Chemical Society, 1968-1990, p.1-24; preparation of allyl silanes: Anderson and Fuchs, 1987, SyntheticCommun. 17:621) and an array of carbon nucleophiles available fromcommercial sources. Additional examples include, trimethylsilyl enolethers, allyltrimethylsilane, E- and Z-crotyltrialkylsilanes,organoaluminum reagents, trialkylstannanes, propargylictrialkylstannanes, 1-(acetoxy)-2-propenyl!trimethylsilane,1-(acetoxy)-2-methyl-2-propenyl!-trimethylsilane, andethyl-2-propenyltrimethyl-silane-1-carbonate. All are efficient carbonnucleophiles in the field of carbon glycosidation reactions (Panek andSparks, 1989, J. Org. Chem. 54:2034, and references therein). The use of1-(acetoxy)-2-methyl-2-propenyl!-trimethylsilane reagent provides accessto terminally oxygen substituted propenyl groups.

Although carbon glycosides can be produced in a few synthetictransformations, they do not necessarily form suitable carbon glycosideswhich could easily be used as alkylating agents for the preparation ofnovel carbohydrate mimics. In one aspect, the present invention providesnovel carbohydrate analogues for the preparation of carbohydratemimetics. Libraries of glycomimetics of complex carbohydrates such as,but not limited, to Sialyl Lewis^(x) (sLe^(x)) tetrasaccharide can beprepared (Rao et al., 1994, The Journal of Biological Chemistry269:19663; Allanson et al., 1994, Tetrahedron Asymmetry 5:2061). One ofthe advantages of having an allylic halide as an alkylating agent is itwould not be prone to E-2 elimination reactions (see, among otherplaces, Lowry and Richardson, Mechanisms and Theory in OrganicChemistry, Second edition, 1981, Harper & Row, New York, p. 530). Amongthe distinct advantages of this type of novel carbon glycoside is theplethora of new chemical entities created by virtue of the invention.

For example, several terminally substituted halogen carbon glycosidesare efficiently obtained from reaction of2-chloromethyl-3-trimethylsilyl-1-propene or2-chloromethyl-3-trimethoxysilyl-1-propene with an activatedcarbohydrate when the reaction is catalyzed by Lewis acid. Thereby, theallylsilanes can undergo a stereochemically controlled axial addition tothe pyranose oxonium ions produced by Lewis acid catalysis and anomericacetates. Benzyl protected carbohydrates result in a stereoselective andefficient route to A-C-glycosides, incorporating an allylic chloride.The use of the per-O-acetylated carbohydrates offers added versatilityby avoiding the hydrogenolysis step required for O-benzyl protectedsugars. Nashed and Anderson, 1982, J. Amer Chem. Soc. 104:7282; Panekand Sparks, 1989, J. Org. Chem. 54:2034.

2-Chloromethyl-3-trimethylsilyl-1-propene and2-chloromethyl-3-trimethoxysilyl-1-propene reagents react with benzylprotected carbohydrates with equal efficiency while per-O-acetylatedcarbohydrates show better results with the2-chloromethyl-3-trimethylsilyl-1-propene reagent. Examples for thecarbon glycoside synthesis as employed by the subject invention areprovided by the instant disclosure, infra. Both the α- and the β-configuration are part of the invention.

Methods for the Generation of Novel Compounds Comprising CarbonGlycosides

The tools and methods of the present invention are focused towards theincorporation of carbohydrates into existing and novel organiccompounds, and combinatorial chemical libraries.

The carbohydrate moieties employed for the generation of such naphthylcompounds and libraries include monomers, dimers, trimers, oligomers,branched or unbranched, linked to a suitable functional group of achemical moiety comprising such functional group. Suitable functionalgroups include, but are not limited to, phenolic, hydroxyl, carboxyl,thiol, amido, and amino groups. In the case a moiety has more than onesuch suitable functional group, one or more such functional groups maybe protected by suitable protecting groups during the coupling reaction.Such protecting groups include lower methyl-, benzyl-, benzoyl-,acetyl-, MOM, MEM, MPM, tBDMS, or TMS groups. After the couplingreaction, the protecting groups may selectively be removed.

In all cases, every molecule comprising at least one suitable functionalgroup can be employed as substrate to react with the activated carbonglycosides/heteroatom glycosides of the present invention.

Protecting Groups

The monomers of the present invention, i.e., the carbohydrates used forthe formation of carbon glycosides, the carbon glycosides, and/or thesubstrates may have groups protecting part of the functional groupswithin the monomer. Suitable protecting groups will depend on thefunctionality and particular chemistry used to generate the novelcompound or combinatorial chemical library. Examples for suitablefunctional protecting groups will be readily apparent to those ofordinary skill in the art, and can be found, among other places, inGreene and Wutz, 1991, Protecting Groups in Organic Synthesis, 2d ed.,John Wiley & Sons, N.Y. Protecting groups typically used when modifyingthe anomeric position of carbohydrates are apparent to one of ordinaryskill in the art. In addition, a plurality of functional groups may beemployed. The C-atom of the carbohydrate used for the formation of thecarbon glycosidic bond can be modified by differential protection offunctional groups, as it will be apparent to those of ordinary skill inthe art. Most preferred protecting groups of the present inventioncomprise benzyl- and acetyl-groups.

Coupling Reactions

Carbon glycoside reagents can be functionalized to be used in a plethoraof chemical reactions in order to form unique compounds. Suitablefunctionalized carbon glycosides can be attached, for example, tophenolic, hydroxyl, carboxyl, thiol, amino, amido, and/or equivalentfunctionality under mild conditions. Activated forms of C-glycosides arealkylhalide, alkenyl halide, or some equivalent activated form. Thecoupling reactions for activated C-glycosides can be performed to formnovel compounds under standard conditions typically used for alkyl andalkenyl chlorides, bromides, iodides, acetates, alcohols, Grignards,etc. In addition, the alkenyl C-glycosides offer routes into Coperearrangements, Claisen rearrangements, and allylic couplings, as theyare readily known by those of ordinary skill in the art and as aredescribed in various examples provided hereinbelow to form novelglycomimetics and unique compounds.

Many named standard reaction conditions using allylic halides parallelthe use of carbon glycosides containing allylic halide functionality toprepare novel compounds and functional groups, including but not limitedto the Alper Reaction, Barbier Reaction, Claisen-Ireland Reaction, CopeRearrangement, Delepine Amine synthesis, Gewald Heterocycle Synthesis,Hiyama-Heathcock Stereoselective Allylation, Stork Radical Cyclization,Trost Cyclopentanation, Weidenhagen Imidazole Synthesis. See, ingeneral, Hassner and Stumer, 1994, "Organic Syntheses Based on NamedReactions and Unnamed Reactions", Tetrahedron Organic Chemistry Series,edts. Baldwin and Magnus, Pergamon, Great Britain.

One of ordinary skill in the art will appreciate that the methods of thepresent invention can be used to incorporate carbohydrate units oranalogues thereof in virtually any naphthyl based structure. In the casewhere a substrate Naphthyl structure comprises more than one suitablefunctional group to react with the functionalized C-glycosides of thepresent invention, these functionalities need not be identical.

Interconversion of Alkenyl Linkers

The alkenyl C-Glycoside compounds described and utilized here are ofparticular utility due to the reactivity of the activated alkenylC-glycoside reagent, and the diverse array of transformations andfunctional group modifications possible around the alkenyl moiety. Thescheme below illustrates some of the more common transformations thatcan be applied to these structures, with the understanding that manyadditional modifications are possible to one of ordinary skill in theart. ##STR29##

As illustrated in the above scheme, the alkenyl moiety is easilyconverted to the saturated substituted alkyl form using standardcatalytic hydrogenation conditions as found in advanced organicchemistry reference textbooks. Treatment of the alkene with 9-BBNfollowed by treatment with basic hydrogen peroxide, or equivalentconditions, provided the primary alcohol derivative. Oxidation of thealcohol using known conditions would provide the carboxylic acid analog.The vicinal diol derivative can be prepared by oxidation with osmiumtetra oxide and NMO or similar bishydroxylation conditions as describedin standard texts on organic chemistry. The ketone derivative can bemade from diol by oxidative cleavage reaction conditions includingperiodate oxidation. Alternately, the ketone can be prepared by directtreatment of the alkene with osmium tetroxide and periodate. The ketonecan then provide the secondary alcohol by reduction with reducing agentssuch as sodium borohydride.

The diol group described above is suited to a variety of O-alkylationand O-acylation reactions, with O-sulfation being a particularly usefulmethod for introducing anionic functionality into the linker. The ketonecan undergo subsequent modifications via wittig reactions as describedto yield the substituted alkene groups, or following reduction bycatalytic hydrogenation, yielding substituted alkyl linker groups.

The common transformations described above are generally known to oneskilled in the art and the methodologies described, and equivalentmethodologies are available in advanced organic chemistry textbooks, andreferences cited therein. For some general references to thesechemistries please see the appropriate Example.

Claisen-Ireland Rearrangement: General Example.

The rearrangement of allyl aromatic ethers to ortho or paraallylaromatic alcohols, or the rearrangement to adjacent positionsrelative to the alkylated phenol moiety, can be accomplished in a twostep format. The first step is to alkylate the phenol, which can be doneusing the procedure herein disclosed or by using the optional procedureused in the following example using sodium methoxide. The result is theo-alkylated napthols.

The alkylated naphthols can then be refluxed in the appropriate solventsuch as dimethylaniline or 1,2-dichlorobenzene for 3-12 hours (dependingon the substrate) to give the Claisen-Ireland product.

Claisen-Ireland Rearrangement: 2-Naphthol Example.

The rearrangement of allyl naphthol ethers to ortho or para allylphenolscan be accomplished in a two step format. The first step is to alkylatethe phenol, which can be done using the procedures herein disclosed.##STR30##

The allylic naphthol is refluxed in dimethylaniline or dichlorobenzenefor 3-12 hours to give the1-(allyl-2,3,4-tri-O-benzyl-alpha-C-Fucoside)-2-naphthol.

Aromatic Nucleophiles: The O-C Migration.

The O to C rearrangement of O-phenyl glycosides has been reported togenerate aromatic C-glycosides (M.H.D. Postema, C-Glycoside Synthesis,CRC Press, page 2-42, c1995, CRC Press, Inc., ISBN 0-8493-9150-4, AnnArbor, Mich., and references therein). Kometani and collaborators (T.Kometani, H. Kondo, Y. Fumimori, Synthesis, 1005, (1988)) have used theO to C migration for the preparation of aromatic C-glycosides. Its usein the preparation of sLex mimics and its use in conjunction with theClaisen-Ireland rearrangement contained in this application is new. Whenthese compounds are exposed to a Lewis acid, the rearrangement takesplace to give the beta-aryl-C-glycoside. Typically, beta isomer is themajor component. In these reactions, the regioselectivity of 2-naphtholsgives the C-glycoside at the 1-position while the 1-naphthols give theC-glycoside at the 2-position. Using this methodology in conjunctionwith the Claisen-Ireland type of rearrangements gives novel compounds ofthe invention.

Example (Kometani, et al): ##STR31## Use and Administration

The compounds of the invention such as various ligands of structuralformula I can be administered to a subject in need thereof to treat thesubject by either prophylactically preventing inflammation or relievingit after it has begun. The ligands are preferably administered with apharmaceutically acceptable carrier, the nature of the carrier differingwith the mode of administration, for example, oral administration,usually using a solid carrier and I.V. administration a liquid saltsolution carrier. The formulation of choice can be accomplished using avariety of excipients including, for example, pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, sodium saccharincellulose, magnesium carbonate, and the like. Oral compositions may betaken in the form of solutions, suspensions, tablets, pills, capsules,sustained release formulations, or powders. Particularly useful is theadministration of the compounds directly in transdermal formulationswith permeation enhancers such as DMSO. Other topical formulations canbe administered to treat dermal inflammation.

A sufficient amount of compound(s) would be administered to bind to asubstantial portion of the selectin expected to cause or actuallycausing the disease, for example, inflammation so that inflammation caneither be prevented or ameliorated. Thus, "treating" as used hereinshall mean preventing or ameliorating the appropriate disease.Typically, the compositions of the instant invention will contain fromless than 1% to about 95% of the active ingredient, preferably about 10%to about 50%. Preferably, between about 10 mg and 50 mg will beadministered to a child and between about 50 mg and 1000 mg will beadministered to an adult. The frequency of administration will bedetermined by the care given based on patient responsiveness.

Other effective dosages can be readily determined by one of ordinaryskill in the art through routine trials establishing dose responsecurves.

When determining the dose of compounds to be administered which blockselectin receptors, it must be kept in mind that one may not wish tocompletely block all of the receptors. In order for a normal healingprocess to proceed, at least some of the white blood cells orneutrophils must be brought into the tissue in the areas where thewound, infection or disease state is occurring. The amount of theligands administered as blocking agents must be adjusted carefully basedon the particular needs of the patient while taking into consideration avariety of factors such as the type of disease that is being treated.

Other modes of administration will also find use with the subjectinvention. For instance, the ligand molecules of the invention can beformulated in suppositories and, in some cases, aerosol and intranasalcompositions. For suppositories, the vehicle composition will includetraditional binders and carriers such as, polyalkylene glycols, ortriglycerides. Such suppositories may be formed from mixtures containingthe active ingredient in the range of about 0.5% to about 10% (w/w),preferably about 1% to about 2%.

Intranasal formulations will usually include vehicles that neither causeirritation to the nasal mucosa nor significantly disturb ciliaryfunction. Diluents such as water, aqueous saline or other knownsubstances can be employed with the subject invention. The nasalformulations may also contain preservatives such as, but not limited to,chlorobutanol and benzalkonium chloride. A surfactant may be present toenhance absorption of the subject proteins by the nasal mucosa.

The compounds of the instant invention may also be administered asinjectables. Typically, injectable compositions are prepared as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid vehicles prior to injection may also be prepared.The preparation may also be emulsified or the active ingredientencapsulated in liposome vehicles.

Compounds of formula I can be mixed with compatible, pharmaceuticallyacceptable excipients. Suitable vehicles are, for example, water,saline, dextrose, glycerol, ethanol, or the like, and combinationsthereof. In addition, if desired, the vehicle may contain minor amountsof auxiliary substances such as wetting or emulsifying agents or pHbuffering agents. Actual methods of preparing such dosage forms areknown, or will be apparent, to those of ordinary skill in the art. See,e.g. Remington's Pharmaceutical Sciences, Mack Publishing Company,Easton, Pa., 17th edition, 1985. The composition or formulation to beadministered will, in any event, contain a quantity of the compoundsadequate to achieve the desired state in the subject being treated.

The various compounds of the present invention can be used by themselvesor in combination with pharmaceutically acceptable excipient materialsas described above. However, the compounds of the invention can be madeas conjugates wherein they are linked in some manner (e.g., via the R¹moiety) to a label. By forming such conjugates, the compounds can act asbiochemical delivery systems for the label so that a site of disease canbe detected.

For instance, carbohydrates can be labelled by a variety of procedures,for example: esterification of hydroxyl bonds to form a structurecapable of complexing directly with a radioisotope or NMR enhancer;reaction of the carbohydrate with amino diacetic acid (IDA) in organicsolvent to form an N-linked glycoside derivative which would be capableof complexing with a radioisotope via the nitrogen and oxygen atoms ofthe IDA group; or coupling of the carbohydrate to amino acids which maybe labelled directly (e.g. cysteine, tyrosine) or labelled via abifunctional chelating agent (e.g., lysine).

Appropriate radioactive atoms would include, for example, technetium 99m(^(99m) Tc), iodine-123 (¹²³ I) or indium-111 (¹¹¹ In) for scintigraphicstudies, or for nuclear magnetic resonance (NMR) imaging (also known asmagnetic resonance imaging, MRI), a label such as gadolinium, manganeseor iron, or a positron-emitting isotope such as iodine-124, fluorine-19,carbon-13, nitrogen-15 or oxygen-17.

EXAMPLES

The compounds of this invention and their preparation can be understoodfurther by the following examples which illustrate some of the processesby which these compounds are prepared. The following examples areprovided so as to provide those of ordinary skill in the art with acomplete disclosure and description of how to make compounds andcompositions of the invention and are not intended to limit the scope ofwhat the inventors regard as their invention. The compounds of thepresent invention can be prepared by methods now known or laterdeveloped. Unless indicated otherwise, parts are parts by weight,temperature is in degrees C, and pressure is at or near atmospheric.

Materials and Methods

Reagents were purchased from commercial suppliers such as PfanstiehlLaboratories, Aldrich Chemical Company or Lancaster Synthsis Ltd. andwere used without further purification unless otherwise indicated.Tetrahydrofuran (THF) and dimethylformamide (DMF) were purchased fromAldrich in sure seal bottles and used as received. All solvents werepurified by using standard methods readily known to those of ordinaryskill in the art unless otherwise indicated.

General Protocol

The reactions set forth below are done generally under a positivepressure of nitrogen or with a drying tube, at ambient temperature(unless otherwise stated), in anhydrous solvents, and the reactionflasks were fitted with rubber septa for the introduction of substratesand reagents via syringe. Glassware was oven dried and/or heat dried.Analytical thin layer chromatography (TLC) was performed on glass-backedsilica gel 60 F 254 plates Analtech (0.25 mm) and eluted with theappropriate solvent ratios (v/v) which are noted where appropriate. Thereactions were assayed by TLC and terminated as judged by theconsumption of starting material.

Visualization of the TLC plates were done with a p-anisaldehyde sprayreagent or phosphomolybdic acid reagent (Aldrich Chemical 20% wt inethanol) and activated with heat. Work-ups were typically done bydoubling the reaction volume with the reaction solvent or extractionsolvent and then washing with the indicated aqueous solutions using 25%by volume of the extraction volume unless otherwise indicated. Productsolutions were dried over anhydrous Na₂ SO₄ prior to filtration andevaporation of the solvents under reduced pressure on a rotaryevaporator and noted as solvents removed in vacuo.

Flash column chromatography (Still et al., 1978, A.J. Org. Chem.43:2923) was done using Baker grade flash silica fel (47-61 mm) and asilica gel: crude material ratio of about 20:1 to 50:1 unless otherwisestated.

Hydrogenolysis can be done at the pressure indicated in the examples, orat ambient pressure.

¹ H-NMR spectra were recorded on a Varian 300 instrument operating at300 MHz and ¹³ C-NMR spectra were recorded on a Varian 300 instrumentoperating at 75 MHz. NMR spectra were obtained as CDCl₃ solutions(reported in ppm), using chloroform as the reference standard (7.25 ppmand 77.00 ppm) or CD₃ OD (3.4 and 4.8 ppm and 49.3 ppm) or internallytetramethylsilane (0.00 ppm) when appropriate. When peak multiplicitiesare reported, the following abbreviations are used: s (singlet), d(doublet), t (triplet), m (multiplet), br (broadened), dd (doublet ofdoublets), dt (doublet of triplets). Coupling constants, when given, arereported in Hertz.

Infrared spectra were recorded on a Perkin-Elmer FT-IR Spectrometer asneat oils, or a CDCl₃ solutions, and are reported in wave numbers(cm⁻¹).

The mass spectra were obtained using LSIMS. All melting points areuncorrected. Microanalyses were carried out by Galbraith Laboratories,Inc., Knoxville, Tenn.

Catalytic Reduction

Catalytic Hydrogenation for the Reduction of an Alkene or Removal of theBenzyl Group.

For a compound containing an alkene, 1.00 mmole equivalent is dissolvedin an appropriate hydrogenation solvent suitable for the compound to bedeprotected. Solvents can include but are not restricted to, methanol,ethyl acetate, ethanol, acetic acid or combinations thereof. Forexample, methanol with a catalytic amount of acetic acid or ethylacetate and methanol can be used as the hydrogenation solvent. 5% or 10%palladium on carbon (1 g for every 50 grams of starting material withthe catalyst wetted with toluene under argon) is evacuated and hydrogengas is added and the process repeated three times. The reaction isshaken or stirred for several hours until the deprotection is complete.The reaction can be done under ambient pressures or can be performedusing a hydrogenation bomb at appropriate pressures (generally 10-50psig). The reaction is terminated by removal of the excess hydrogen gas,flushing the reaction vessil with an inert atmosphere and then filteringthe contents through Celite to remove the catalyst and washing thecatalyst with 30% methanol in chloroform or appropriate solvent system.Concentration in vacuo afforded the desired compound. The product can bepurified by column chromatography using Baker grade fresh silica gel(47-61 mm) and a suitable solvent system. For example, 10% ethyl acetatein hexanes and then with 30% ethyl acetate in hexanes. The silica gel iseluted with methanol and checked by TLC for any product material. Thesolvents are removed in vacuo and the product dried under vacuum. Thedesired product is recovered.

General references on applicable transformations can be found, amongother places, in:

R. C. Larock, Comprehensive Organic Transformations, ISBN 0-89573-710-8,1989, VCH Publishers, Inc. 220 East 23rd Street, Suite 909, New York,N.Y. 10010.

Jerry March, Advanced Organic Chemistry, 3rd Edition, c1985, ISBN0-471-88841-9, John Wiley & Sons Publishers, New York USA.

H. O. House, Modern Synthetic Reactions, c1972, ISBN 0-8053-4501-9, TheBenjamin/Cummings Publishing Company, Menlo Park, Calif., USA.

F. A. Carey and R. J. Sundberg, Advanced Organic Chemistry Parts A & B,2nd Edition, c1983, ISBN 0-306-41199-7, Plenum Press, a division ofPlenum Publishing Corporation, 233 Spring Street, New York, N.Y. 10013,USA.

9-BBN

For a compound containing an alkene, (1.00 mmole equiv.) is dissolved inan appropriate solvent suitable for the compound to be reduced with9-BBN (9-borabicyclo 3.3.1!nonane) or equivalent. Solvents can includebut are not restricted to, tetrahydrofuran (THF), hexanes and diethylether, or combinations thereof. To a stirred solution of the olefin,(1.00 mmole equiv.) in THF (0.5M) at 0° C. is added 9-BBN pre-dissolvedin THF (1.00 mmole equiv.) is carefully added. The reaction contents arestirred at 0° C. and the cooling bath (water/ice) is allowed to melt.The reaction is allowed to stir at ambient temperature for 8 hours oruntil the reaction is complete via analysis by TLC. The reaction isterminated by the careful addition acetone and stirred for 1 hour atroom temperature. The reaction contents are cooled to 0° C. and then1.0M NaOH and 30% H₂ O₂ are added to decompose the alkyl borate. Thecontents are stirred until the alkyl borate decomposes to the carbonol.An extraction solvent such as chloroform or ethyl acetate is added andthe heterogeneous layers are separated and the organic phase is washedwith 1.0M hydrochloric acid, water and brine. The washed product isdried over anhydrous sodium sulfate and filtered. The product can bepurified by column chromatography using Baker grade flash silica gel(47-61 mm) and a suitable solvent system. For example, 10% ethyl acetatein hexanes and then with 30% ethyl acetate in hexanes or appropriatesolvent mixtures dependant upon the particular substrate used. Thesilica gel is eluted with methanol and checked by TLC for any productmaterial. The solvents are removed in vacuo and the product dried undervacuum the desired product is recovered.

General references on applicable transformations can be found, amongother places, in:

R. C. Larock, Comprehensive Organic Transformations, ISBN 0-89573-710-8,1989, VCH Publishers, Inc. 220 East 23rd Street, Suite 909, New York,N.Y. 10010.

Jerry March, Advanced Organic Chemistry, 3rd Edition, c1985, ISBN0-471-88841-9, John Wiley & Sons Publishers, New York USA.

H. O. House, Modern Synthetic Reactions, c1972, ISBN 0-8053-4501-9, TheBenjamin/Cummings Publishing Company, Menlo Park, Calif., USA.

F. A. Carey and R. J. Sundberg, Advanced Organic Chemistry Parts A & B,2nd Edition, c1983, ISBN 0-306-41199-7, Plenum Press, a division ofPlenum Publishing Corporation, 233 Spring Street, New York, N.Y. 10013,USA.

Oxidation

General references on applicable transformations can be found, amongother places in:

M. Hudlicky, Oxidations in Organic Chemistry, ACS Monograph #186, c1990,ISBN 0-8412-1781-5, Published by The American Chemical Society,Washington, D.C.

R. C. Larock, Comprehensive Organic Transformations, ISBN 0-89573-710-8,1989, VCH Publishers, Inc. 220 East 23rd Street, Suite 909, New York,N.Y. 10010.

Jerry March, Advanced Organic Chemistry, 3rd Edition, c1985, ISBN0-471-88841-9, John Wiley & Sons Publishers, New York USA.

H. O. House, Modern Synthetic Reactions, c1972, ISBN 0-8053-4501-9, TheBenjamin/Cummings Publishing Company, Menlo Park, Calif., USA.

F. A. Carey and R. J. Sundberg, Advanced Organic Chemistry Parts A & B,2nd Edition, c1983, ISBN 0-306-41199-7, Plenum Press, a division ofPlenum Publishing Corporation, 233 Spring Street, New York, N.Y. 10013,USA.

Bis-Hydroxylation

Bishydroxylation of the olefin via Oxidation with osmium tetroxide. To astirred solution of the olefin, (1.00 mmole equiv.) in 1% water inacetone (0.5M) at 0° C. is added osmium tetroxide pre-dissolved inacetone (0.01 mmole equiv.) and N-methylmorpholine-N-oxide (2.00 mmoleequiv.) is carefully added as a solid. The reaction contents are stirredat 0° C. and the cooling bath (water/ice) is allowed to melt. Thereaction is allowed to stir at ambient temperature for 18 hours or untilthe reaction is complete via analysis by TLC. The reaction can beassayed by TLC or an aliquot of the reaction acetate. The aliquot ischecked by ¹ H-NMR in CDCl₃. The reaction is terminated by the carefuladdition of sodium bisulfite (contains a mixture of NaHSO₃ and Na₂ S₂O₅), stirred for 1 hour at room temperature and then water. Anextraction solvent such as chloroform is added and the heterogeneouslayers are separated and the organic phase is washed with 1.0Mhydrochloric acid, water and brine. The washed product is dried overanhydrous sodium sulfate and filtered. The product can be purified bycolumn chromatography using Baker grade flash silica gel (47-61 mm) anda suitable solvent system. For example, 10% ethyl acetate in hexanes andthen with 30% ethyl acetate in hexanes. The silica gel is eluted withmethanol and checked by TLC for any product material. The solvents areremoved in vacuo and the product dried under vacuum the desired productis recovered.

General references on applicable transformations can be found, amongother places, in:

R. C. Larock, Comprehensive Organic Transformations, ISBN 0-89573-710-8,1989, VCH Publishers, Inc. 220 East 23rd Street, Suite 909, New York,N.Y. 10010.

Jerry March, Advanced Organic Chemistry, 3rd Edition, c1985, ISBN0-471-88841-9, John Wiley & Sons Publishers, New York USA.

H. O. House, Modern Synthetic Reactions, c1972, ISBN 0-8053-4501-9, TheBenjamin/Cummings Publishing Company, Menlo Park, Calif., USA.

F. A. Carey and R. J. Sundberg, Advanced Organic Chemistry Parts A & B,2nd Edition, c1983, ISBN 0-306-41199-7, Plenum Press, a division ofPlenum Publishing Corporation, 233 Spring Street, New York, N.Y. 10013,USA.

Sulfation

Sulfation of hydroxyl functionalities. In a preferred embodiment, thehydroxy groups of the target molecule are sulfated under standardconditions: For example, to a solution of 0.35 g 3,4-di-C-fucosylcaffeic acid (0.603 mmole, 1 mmole equiv.) in 7.2 mL pyridine was added1.15 g sulfur trioxide pyridine complex (7.24 mmole, 12 mmole equiv.)and the reaction was stirred at ambient temperature for 12 hours. Thereaction was complete as assayed by TLC at CHCl₃ :MeOH:H₂ O 10:10:1(v/v) as assay conditions. To the mixture was added methanol and thesolution was stirred for 1 hour. All of the solvents were evaporated andthe residue was chromatographed on Bakerbond Octadecyl (40 μm) silicagel and eluted with water and 10% methanol in water. The combinedfractions were subjected to sodium ion exchange resin for the exchangeof residual ionic salts for sodium ions. Lyophilization providedpersulfated 3,4-di-C-fucosyl sodium cafeate.

As an additional alternative method: To a solution of the alcohol(s)groups to be sulfated from the products of the invention (1.00 mmoleequiv.) in anhydrous pyridine or diemthylformamide (0.2M) at ambienttemperature was added sulfur trioxide pyridine complex of the sulfurtrioxide pyridine complex polymer bound Graf, W. chem. Ind. 1987, 232.!(10 mmole equiv.). The reaction contents were stirred at ambienttemperature for 8 hours. The reaction was quenched using sodiumcarbonate and removing the solvents by lyophilization and the resultingmaterial was subjected to sodium ion exchange resin for the exchange ofresidual ionic salts for sodium ions. Concentration in vacuo affords thesulfated materials.

The experimental procedures described can be applied to make and modifythe following exemplified novel products.

General references on applicable transformations can be found, amongother places, in:

R. C. Larock, Comprehensive Organic Transformations, ISBN 0-89573-710-8,1989, VCH Publishers, Inc. 220 East 23rd Street, Suite 909, New York,N.Y. 10010.

G. A. Olah, et al, Synthesis, 59, (1979), G. A. Olah, et al, Synthesis,984, (1979), Y. Hamada, T. Shiori, Chem. Pharm. Bull. 30, 1921, (1982).

Ketone Formation

Oxidation of the alkene to the ketone via catalytic oxidation withosmium tetroxide sodium periodate. To a stirred solution of the olefin,(1.00 mmole equiv.) in 1% water in acetone (0.5M) at 0° C. is addedosmium tetroxide pre-dissolved in acetone (0.01 mmole equiv.) and sodiumperiodate (2.00 mmole equiv.) is carefully added as a solid. Thereaction contents are stirred at 0° C. and the cooling bath (water/ice)is allowed to melt and the reaction allowed to stir at ambienttemperature for 18 hours or until the reaction is complete via analysisby TLC. The reaction can be assayed by TLC or an aliquot of the reactioncontents is removed, quenched into aqueous sodium metasulfite andextracted with ethyl acetate. The aliquot is checked by ¹ H-NMR. Thereaction is terminated by the careful addition of sodium bisulfite(contains a mixture of NaHSO₃ and NA₂ S₂ O₅), stirred for 1 hour at roomtemperature and then water. An extraction solvent such as chloroform isadded and the hydrochloric acid, water and brine. The washed product isdried over anhydrous sodium sulfate and filtered. The product can bepurified by column chromatography using Baker grade flash silica gel(47-61 mm) and a suitable solvent system. For example, 10% ethyl acetatein hexanes and then with 30% ethyl acetate in hexanes. The silica gel iseluted with methanol and checked by TLC for any product material. Thesolvents are removed in vacuo and the product dried under vacuum. Thedesired product is recovered.

General references on applicable transformations can be found, amongother places, in:

M. Hudlicky, Oxidations in Organic Chemistry, ACS Monograph #186, c1990,ISBN 0-8412-1781-5, Published by The American Chemical Society,Washington, D.C.

R. Pappo, D. S. Allen Jr., R. U. Lemieux, W. S. Johnson, J. Org. Chem.,21, 478, (1956).

R. C. Larock, Comprehensive Organic Transformations, ISBN 0-89573-710-8,1989, VCH Publishers, Inc. 220 East 23rd Street, Suite 909, New York,N.Y. 10010.

Jerry March, Advanced Organic Chemistry, 3rd Edition, c1985, ISBN0-471-88841-9, John Wiley & Sons Publishers, New York USA.

H. O. House, Modern Synthetic Reactions, c1972, ISBN 0-8053-4501-9, TheBenjamin/Cummings Publishing Company, Menlo Park, Calif., USA.

F. A. Carey and R. J. Sundberg, Advanced Organic Chemistry Parts A & B,2nd Edition, c1983, ISBN 0-306-41199-7, Plenum Press, a division ofPlenum Publishing Corporation, 233 Spring Street, New York, N.Y. 10013,USA.

Wittig (Ester)

The use of a Wittig reagent for the conversion of a ketone to an alpha,beta unsaturated ester can be found in the additional references:

R. C. Larock, Comprehensive Organic Transformations, ISBN 0-89573-710-8,1989, VCH Publishers, Inc., 220 East 23rd Street, Suite 909, New York,N.Y. 10010

Jerry March, Advanced Organic Chemistry, 3rd Edition, c1985, ISBN0-471-88841-9, John Wiley & Sons Publishers, New York, USA.

H. O. House, Modern Synthetic Reactions, c1972, ISBN 0-8053-4501-9, TheBenjamin/Cummings Publishing Company, Menlo Park, Calif., USA.

F. A. Carey and R. J. Sundberg, Advanced Organic Chemistry Parts A & B,2nd Edition, c1983, ISBN 0-306-41199-7, Plenum Press, a division ofPlenum Publishing Corporation, 233 Spring Street, New York, N.Y. 10013,USA.

Wittig (Alkene)

The use of a radiolabelled Wittig reagent for the conversion of a ketoneto an alkene can be found in Bioorg. Chem. 19, 327, (1991) or J. Amer.Chem Soc. 111, 3740, (1989). Additional references can be found onapplicable transformations in:

R. C. Larock, Comprehensive Organic Transformations, ISBN 0-89573-710-8,1989, VCH Publishers, Inc., 220 East 23rd Street, Suite 909, New York,N.Y. 10010.

Jerry March, Advanced Organic Chemistry, 3rd Edition, c1985, ISBN0-471-88841-9, John Wiley & Sons Publishers, New York, USA.

H. O. House, Modern Synthetic Reactions, c1972, ISBN 0-8053-4501-9, TheBenjamin/Cummings Publishing Company, Menlo Park, Calif., USA.

F. A. Carey and R. J. Sundberg, Advanced Organic Chemistry Parts A & B,2nd Edition, c1983, ISBN 0-306-41199-7, Plenum Press, a division ofPlenum Publishing Corporation, 233 Spring Street, New York, N.Y. 10013,USA.

Sodium Borohydride Reduction

For a compound containing a ketone, (1.00 mmole equiv.) is dissolved inan appropriate solvent suitable for the compound to be reduced withsodium borohydride or equivalent. Solvents can include but notrestricted to, tetrahydrofuran (THF), hexanes and diethyl ether, orcombinations thereof. To a stirred solution of the ketone, (1.00 mmoleequiv.) in THF (0.5M) at 0° C. is added sodium borohydridepre-dissolved/suspended in THF (1.00 mmole equiv.) is carefully added.The reaction contents are stirred at 0° C. and the cooling bath(water/ice) is allowed to melt and the reaction allowed to stir atambient temperature for 8 hours or until the reaction is complete viaanalysis by tlc. The reaction is terminated by the careful addition ofacetone and stirred for 1 hour at room temperature. Water is carefullyadded and the reaction contents stirred to decompose the alkyl borate.The contents are stirred until the alkyl borate decomposes to thecarbonol. An extraction solvent such as chloroform or ethyl acetate isadded and the heterogeneous layers are separated and the organic phaseis washed with 1.0M hydrochloric acid, water and brine. The washedproduct is dried over anhydrous sodium sulfate and filtered. The productcan be purified by column chromatography using Baker grade flash silicagel (47-61 mm) and a suitable solvent system. For example, 10% ethylacetate in hexanes and then with 30% ethyl acetate in hexanes orappropriate solvent mixtures dependent upon the particular substrateused. The silica gel is eluted with methanol and checked by TLC for anyproduct material. The solvents are removed in vacuo and the productdried under vacuum until the desired product is recovered.

General references on applicable transformations can be found, amongother places, in:

R. C. Larock, Comprehensive Organic Transformations, ISBN 0-89573-710-8,1989, VCH Publishers, Inc., 220 East 23rd Street, Suite 909, New York,N.Y. 10010.

Jerry March, Advanced Organic Chemistry, 3rd Edition, c1985, ISBN0-471-88841-9, John Wiley & Sons Publishers, New York, USA.

H. O. House, Modern Synthetic Reactions, c1972, ISBN 0-8053-4501-9, TheBenjamin/Cummings Publishing Company, Menlo Park, Calif., USA.

F. A. Carey and R. J. Sundberg, Advanced Organic Chemistry Parts A & B,2nd Edition, c1983, ISBN 0-306-41199-7, Plenum Press, a division ofPlenum Publishing Corporation, 233 Spring Street, New York, N.Y. 10013,USA.

EXAMPLE 1 Generation of Compounds: General Reaction

General Experimental Procedures:

One of ordinary skill in the art will appreciate and understand thefollowing general experimentals as they are used in the art to preparenovel compounds from the invention. The mmole equivalents refers to thereaction substrate to be functionalized by the reaction with the carbonglycoside reagent per position to be alkylated. Additional functionalgroup transformations can be accomplished by the skilled artisan usingstandard reaction conditions. For example, the transformation of allylichalides into allylic amines can be via the allylic azide with reductionof the azide to the amine with triphenylphosphine in water. The amine isthen available for amide bond formation. ##STR32##

Alkylation conditions using Sodium hydride and an Aliphatic Alcohol. Toa mechanically stirred solution of sodium hydride (3.00 mmole equiv.Note: the sodium hydride is washed three times with hexanes prior touse.) in THF (slurry) at ambient temperature is added an aliphaticalcohol (1.00 mmole equiv.) dropwise in a minimum of anhydroustetrahydrofuran. Tetrabutylammonium iodide (0.10 mmole equiv.) is addedand the reaction contents are stirred at room temperature (slightwarming to above room temperature is sometimes needed for the initiationof the reaction) for 60 minutes in order to minimize the rate of gasevolution. The reaction contents are warmed for a period of 2 hours;carefully watching for the evolution of hydrogen. The reaction contentsare stirred using a mechanical stirrer while being gently refluxed for aperiod of 1.5 hours. A benzyl protected carbon glycoside reagent to beused (1.50 mmole equiv.) is slowly added dropwise in anhydroustetrahydrofuran (total reaction concentration of 0.2 to 0.5M) over aperiod of 1-2 hours and stirred for 4 hours. An aliquot of the reactioncontents is removed and quenched into 1.0M HCl and extracted with ethylacetate; the TLC conditions used are 5% methanol in chloroform (v/v.!The reaction is then diluted with tolune and terminated by the carefuladdition of 50% methanol in toluene at 0° C. to consume the residualsodium hydride, followed by acidification of 1.0M hydrochloric aciduntil the pH is about 2. The reaction contents are diluted with ethylacetate. The heterogeneous layers are separated and the organic phase iswashed twice with portions of 1.0M hydrochloric acid, saturated sodiumthiosulfate and brine. The product can be purified by columnchromatography using Baker grade flash silica gel (47-61 mm) and asuitable solvent system. For example, 10% ethyl acetate in hexanes andthen with 30% ethyl acetate in hexanes. The silica gel is eluted withmethanol and checked by TLC for any product material. The solvents areremoved in vacuo and the product dried under vacuum. The desired productis recovered.

General alkylation conditions using Cesium Carbonate as a mild base forthe alkylation of thiols, amines, carboxylic acids and the like. To astirred solution of thiols, phenols, amines, carboxylic acids and thelike (1.00 mmole equiv.) in DMF or acetone (0.5M) is added cesiumcarbonate (3.00 mmole equiv.) and the Carbon-glycoside reagent (1.50mmole equiv.). The reaction is stirred at room temperature for 12 hours.The reaction is assayed by TLC. The TLC conditions are usually 30% ethylacetate in hexanes (v/v). The reaction contents are diluted with ethylacetate and then poured into cold water. The organic layer is washedtwice with water and then brine. The product is dried over anhydroussodium sulfate and filtered to remove the drying agent. The solvent isremoved in vacuo. The product can be purified by column chromatographyusing Baker grade flash silica gel (47-61 mm) and a suitable solventsystem. For example, 10% ethyl acetate in hexanes and then with 30%ethyl acetate in hexanes. The silical gel is eluted with methanol andchecked by TLC for any product material. The solvents are removed invacuo and the product dried under vacuum. The desired product isrecovered.

Removal of the acetyl protecting groups from the acetylated carbonglycosides. To a solution of the acetyl protected compounds (1.00 mmoleequiv.) in methanol (0.5M) is added cesium carbonate or sodium methoxide(5.00 mmole equiv.) and water (catalytic) and the contents stirred for48 hours. The reaction is quenched with 1.0M HCl until the pH isapproximately 1. The product is extracted with an appropriate extractionsolvent such as chloroform and the organic layer is washed with water.The product is dried over anhydrous sodium sulfate and filtered toremove the drying agent. The product can be purified by columnchromatography using Baker grade flash silica gel (47-61 mm) and asuitable solvent system. For example, 10% ethyl acetate in hexanes andthen with 30% ethyl acetate in hexanes. The silica gel is eluted withmethanol and checked by TLC for any product material. The solvents areremoved in vacuo and the product dried under vacuum. The desired productis recovered.

EXAMPLE 2

Preparation of 1-Deoxy-1-α-Iodoethyl-2,3,4-Tri-O-Acetyl-L-Fucose (2)##STR33##

A solution of tetraacetylated L-fucose (9.0 g, 27.1 mmole) inacetonitrile (100 mmole) was stirred with one teaspoon of powderedmolecular sieves (4 Å) for 30 minutes. This was mixed withallyltrimethylsilane (17.6 ml) followed by boron-trifluoride etherate(17.6 ml) solution in one portion. This reaction mixture was stirred atroom temperature for three days. Most of volatiles were removed in vacuoand diluted with a 1:1 mixture of saturated sodium bicarbonate and brinesolutions (100 ml). This was extracted with ethyl acetate (2×200 ml) andthe combined organic extracts were washed with saturated sodiumbicarbonate and brine solutions, dried over sodium sulfate, filtered andconcentrated. The crude product was purified on a silica gel column(hexane: ethyl acetate, 3:1) to provide allylfucose 7.97₉, 94%).

A solution of allylfucose (5.1 g, 16.3 mmole) in a mixture ofdichloromethane (80 ml) and methanol (20 ml) at -78° C. was bubbled withozone until the blue color persisted. Excess ozone was removed bypassing oxygen through the solution. This was mixed with dimethylsulfide(10 ml) and stirred at -78° C. to room temperature overnight. All thevolatiles were removed in vacuo and dissolved in methanol (50 ml). At-78° C., this was mixed with sodium borohydride (620 mg) and warmed upto 0° C. and stirred for one hour. This is poured into water (50 ml) andextracted with ethyl acetate (2×100 ml). The organic extracts werewashed with brine, dried over sodium sulfate, filtered and concentrated.The crude product was purified on a silica gel column (hexane: ethylacetate, 1:1) to provide the alcohol (1.95 g, 38% yield). A much higheryield (90%) of the alcohol is obtained if sodium triacetoxyborohydrideis used instead of sodium borohydride.

A solution of the above alcohol in pyridine (10 ml) andp-toluenesulfonylchloride (1.75 g) was stirred at 0° C. for 14 hours.This mixture was quenched with water (20 ml) and stirred for 20 minutes.This mixture was acidified with 6N of hydrochloride solution andextracted with ethyl acetate (2×100 ml). The combined organic extractswere then washed with water (100 ml), saturated sodium bicarbonate (30ml), brine (30 ml), dried over sodium sulfate, filtered and concentratedto provide the crude tosylate. This crude product was heated with sodiumiodide (3 g) in acetone (40 ml) at 450° C. for 2 hours. After cooling,the mixture was evaporated and the residue worked up with water (50 ml)and ethyl acetate (200 ml). The crude mixture was purified on a silicagel column (hexane ethyl acetate, 2:1) to provide the iodide 2 (2.9 g,76%).

EXAMPLE 3

Synthesis of "Activated" Carbon Glycoside Building Blocks

A preferred method for the synthesis of terminally substituted halogencarbon glycosides comprises a chemical reaction of2-chloromethyl-3-trimethylsilyl-1-propene (SAF Bulk Chemicals), or2-chloromethyl-3-trimethoxysilyl-1-propene (SAF Bulk Chemicals), or2-chloromethyl-3-trimethoxysilyl-1-propene by Gelest Inc. (U.S. Pat. No.3,696,138), with an activated carbohydrate and a Lewis acid, whereby theallylsilanes undergo addition to the pyranose oxonium ions produced byLewis acid catalysis for example with anomeric acetates. Nashed andAnderson, 1982, Amer, Chem. Soc. 104:7282; Panek and Sparks, 1989, J.Org. Chem, 54:2034. Other electrophilic sugars can be employed as statedearlier (Postema, 1995, supra). It will be apparent to those of ordinaryskill in the art, that also other functional groups at the C-1 positioncan be used to convert the anomeric hydroxyl functionality into anappropriate leaving group at the C-1 position which can be used toconvert the anomeric hydroxyl functionality into an appropriate leavinggroup to form the oxonium ion. 2-chloromethyl-3-trimethylsilyl-1-propeneand 2-chloromethyl-3-trimethoxysilyl-1-propene exhibit about the sameefficiency on the benzyl protected carbohydrates, while at least underthe specific reaction conditions employed the per-O-acetylatedcarbohydrates used the 2-chloromethyl-3-trimethylsilyl-1-propenereagent.

The Carbon glycoside Formation with Benzyl Protected Sugar.

2-Chloromethyl-3-(tri-O-benzylα-L-C-fucopyranoside)-1-propene (3)

To a stirred solution of 500 g of 2,3,4-tri-O-benzyl-L-fucopyranose(1.15 mole) (Pfanstiehl, Inc.) in 500 ml 1,2-dichloroethane or 550 mlTHF, 240 ml acetic anhydride, 135 ml pyridine was added, and the mixturestirred at room temperature for 22 hours. Subsequently, the reaction wasdiluted with ethyl acetate, washed with water, saturated with sodiumbicarbonate and again with water. The solvents were moved in vacuo andazeotroped with toluene. The white solid was placed on a vacuum line,which afforded 542 g (99%) of1-O-Acetyl-2,3,4-tri-O-benzyl-L-fucopyranose as a colorless crystallinesolid in a mixture of anomeric acetates, mp=86°-87.5° C. 564.35 g1-O-Acetyl-2,3,4-tri-O-benzyl-L-fucopyranose (1.19 moles, 1.00 eq.) and214.6 ml 2-chloromethyl-3-trimethylsilyl-1-propene (1.19 mole, 1.00 eq.)were dissolved in 1.2 l acetonitrile (HPLC grade) and cooled toapproximately 0° C. using an ice-water bath. Subsequently, 11.46 mltrimethylsilyltrifluoromethane sulfonate (59.28 mmoles, 0.05 eq.) wascarefully added, the ice-water bath was allowed to melt and the reactionslowly warmed to room temperature (18 hours). Completion of thetransformation from starting material to product was indicated by TLC.The reaction was terminated by pouring the reaction contents onto 1 l ofice-water, followed by adjustment to room temperature. The resultingmixture was extracted with 1 l ethyl acetate and the organic phase thenwashed with 1.0N aqueous sodium hydroxide and brine. The organic phasewas dried over anhdyrous magnesium sulfate, filtered, and the solventwas removed in vacuo, which afforded 600.5 g of2-Chloromethyl-3-(tri-O-benzyl-α-L--C-fucopyranoside)-1-propene (1) aslight yellow solid. The product was purified either by crystallizationin methanol at 0° C., or by column chromatography using Baker gradeflash silica gel (47-61 mm) (ratio of 20 to 1), followed by elution with5 to 10% ethyl acetate in hexanes giving a white solid (98%), mp 47°-49°C. In low scale reactions, the α- to β- ratios of the C-glycosidationreaction, was >10:1 in favor of the α-fucose derivative (Cha, et al.,1982, J. Am. Chem. Soc. 104:4976). The higher the reaction scale, theless β isomer could be observed; at scales in the multi-gram levels,sometimes only trace amounts.

The results of this study are complementary to related C-glycosideformation reactions for pyranosides using an allylic silane and anactivated glycal. In another preferred embodiement, the Finkelsteinexchange of the chloride for iodide (97% yield) or bromide (95% yield)was performed under standard conditions such as NaI in refluxing acetoneor LiBr in refluxing THF. The course of the reaction was monitored byTLC or NMR techniques. Under certain conditions, bromide appears to bemore stable than the corresponding iodide and seems to result in higheryielding alkylations.

This preferred method of forming C-glycosides with the benzyl protectedsugar, 1-O-acetyl-2,3,4-tri-O-benzyl-L-fucopyranoside is reflected inthe following scheme: ##STR34##

The following are typical scales of this reaction: Starting materialAcetate/2-chloromethyl-3-trimethylsilyl-1-propene(310 g/118 ml), Productobtained (331 g); Acetate/2-chloromethyl-3-trimethylsilyl-1-propene(380g/144 ml), Product obtained (400 g).

An alternate procedure starting from the anomeric hydroxyl can be doneas follows: To a solution of 20 g tri-O-benzyl-L-fucopyranose (46.03mmole, 1.00 mmole equiv.) in 200 ml anhydrous acetonitrile 30.0 g2-chloromethyl-3-trimethylsilyl-1-propene (184.34 mmole, 4.00 mmoleequiv.) at 0° C. 10.24 g trimethylsilane trifluoromethane sulfonic acid(46.03 mmol, 1.00 mmole equiv.) was added dropwise in 30 ml anhydrousacetonitrile (overall reaction concentration 0.2M) and the reactioncontents stirred at 0° C. for 30 minutes. After 30 minutes, the reactionwas diluted with 230 ml ethyl acetate and terminated by pouring thecontents slowly into saturated sodium bicarbonate. The heterogenouslayers were separated and the organic phase was washed twice withportions of water, 1.0M hydrochloric acid and brine. The crude productwas dried over anhydrous sodium sulfate, filtered and plugged through asmall pad of silica gel. The solvent was removed in vacuo which affordedan oil that was chromatographed on Baker grade flash silica gel (47-61mm; ratio of 50 to 1) and eluted with 5 to 10% ethyl acetate in hexanes.Concentration in vacuo afforded 20.01 g of2-chloromethyl-3-(tri-O-benzyl-α-L-C-fucopyranoside)-1-propene (85%).MW=507, α!D: -27.37, C=0.95 in CHCl₃. A second product,α-L-2,3,4-tri-O-benzyl-fucopyranose-α-L-2,3,4-tri-O-benzyl-fucopyranose.

The product gave the following analytical data:

Reaction yield: 91%, mp=47°-49° C. ¹ H-NMR (CDCl₃) δ 7.20-7.50 (m, 15H,aromatics), 5.2 (d, J=47.9 Hz, 2H, terminal vinyl), 4.50-4.90 (complexmultiplet, 6H, benzylic), 4.25 (p, 1H, H-1), 4.10 (s, 2H, --CH₂ Cl),3.90 (m, 1H), 3.75 (s, 1H), 2.50 (m, 2H), 1.25 (d, 3H). ¹³ C-NMR (CDCl₃)δ 142.68 alkene (e), 138.62 aromatic (e), 138.29 aromatic (e), 138.11aromatic (e), 128.17 aromatic (o), 127.86 aromatic (o), 127.45 aromatic(o), 127.34 aromatic (o), 116.28 alkene (e), 76.58 (o), 75.95 (o), 73.24(e), 72.97 (e), 68.33 (o), 48.23 --CH₂ Cl (e), 30.30 allylic (e), 15.38fucose methyl (o). Mass Spec. (LSIMS with mNBA) 505.1/507.3. AnalyticalCalculated for CH₃₁ H₃₅ ClO₄ : C, 73.43; H, 6.96. Found: C, 73.16; H.7.12.

EXAMPLE 4

2-Chloromethyl-3-(tetra-O-benzyl-α-L-C-glucopyranocide)1-propene (4)

In another preferred embodiment,1-O-acetyl-2,3,4,6-tetra-O-benzyl-D-glucopyranose was subjected to thesame reaction conditions as have been described for L-fucopyranoside,resulting in the α-C-glycosides of glucose (91% mp=79°-81° C.), asreflected below: ##STR35##

In general, the reagent ratios for the remaining per-O-acetylatedcarbohydrates were for example:1,2,3,4,6-penta-O-acetyl-D-galactopyranoside (1.00 mmole equiv.) and2-chloromethyl-3-trimethylsilyl-1-propene (2.00 mmole equiv.) weredissolved in acetonitrile (1.3M). Boron trifluoride etherate (2.00 mmoleequiv.) and trimethylsilyltrifluoromethane sulfonate (0.40 mmole equiv.)were carefully added neat at room temperature. The reaction was refluxedfor 6 hours and worked up as described. TLC 30% ethyl acetate inhexanes.

The glucose product (4) gave the following analytical data:

Reaction yield: 91%, mp=79°-81° C. ¹ H-NMR (CDCl₃) δ 7.10-7.40 (20H),5.1 (d, J=41.3 Hz, 2H, terminal vinyl), 4.96 (d, 10.87 Hz, 1H), 4.82 (d,10.87 Hz, 1H), 4.82, (d, J=10.56 Hz, 1H), 4.63 (d, J=12.15 Hz, 1H), 4.44(d, J=12.15 Hz, 1H), 4.45 (d, J=1.56 Hz, 1H), 4.67 (q, J=11.6 Hz, 2H),4.24 (p, J=5.07 Hz, 1H, H-1), 4.12 (s, 2H), 3.68 (m, 6H, ring), 2.65 (m,2H). ¹³ C-NMR (CDCl₃) δ 142.32 alkene (e), 138.68 (e), 138.08 (e),137.93 (e), 128.5 (o), 128.0 (o), 127.8 (o), 127.5 (o), 116.95 alkene(e), 82.31 ring (o), 79.85 ring (o), 77.91 ring (o), 75.56 (e), 75.16(e), 73.46 (e), 73.19 (e), 72.80 ring (o), 71.31 ring (o), 68.79CH₂ ring(e), 48.15 CH₂ Cl allylic (e), 27.98 allylic (e). Mass Spec. (LSIMS withmNBA and NaOAc) 635.2 (MNa⁺). Analytical Calculated for C₃₈ H₄₁ ClO₅ :C, 74.43; H, 6.74. Found: C, 74.62; H, 6.92.

EXAMPLE 5

2-Chloromethyl-3-(2,3,4,6-tetra-O-benzyl-D-galactopyranoside)-1-propane(5)

In another preferred embodiment,1-O-acetyl-2,3,4,6-tetra-O-benzyl-D-galactopyranose were subjected tothe same reaction conditions as have been described forL-fucopyranoside, resulting in the α-C-glycosides of galactose (5) (84%,oil), as reflected below: ##STR36##

The product gave the following analytical data:

Reaction yield: 84%, and the compound isolated as an oil. ¹ H-NMR(CDCl₃) δ, 7.25 (m. 20H), 5.16 (d, J=37.54 Hz, 2H), 4.85-4.50(overlapping benzylic patterns, 6H), 4.26 (p, 3.85 Hz, 1H, H-1), 4.16(s, 2H), 4.09 (m, 2H), 3.88 (m, 2H), 3.79 (dd, J=4.88 Hz, 1H), 2.59 (m,2H). ¹³ C-NMR (CDCl₃) δ 143.32 alkene (e), 139.21 (e), 139.09 (e),138.90 (e), 138.83 (e), 128.5 (o), 128.0 (o), 127.8 (o), 127.5 (o),117.22 alkene (e), 77.32 ring (o), 74.89 ring (o), 74.00 (e), 73.88 (e),73.83 (e), 73.69 (e), 72.72 (o), 68.19 (e), 49.09 (e), 28.98 allylic(e). Mass Spec. (LSIMS with mNBA and NaOAc) 635.3 (MNa⁺). AnalyticalCalculated for C₃₈ H₄₁ ClO₅ : C, 74.43; H, 6.74. Found: C, 74.31; H,6.87.

EXAMPLE 6

2-Iodomethyl-3-(2,3,4,-tri-O-benzyl-α-L--C-fucopyranoside)-1-propene (6)

331 grams of2-chloromethyl-3-(tri-O-benzyl-α-L-C-fucopyranoside)-1-propene (653mmole, 1 mmole equiv.) was added to a stirred suspension of 480 g NaI(3222 mmole, 5 mmole equiv.) in 3 l acetone; the reaction was heated toreflux for 3 hours and then allowed to cool to room temperature.Completion of the reaction was monitored by TLC assay. The TLCconditions used were 10% ethyl acetate in hexanes (v/v). The reactionwas complete when the product Rf was slightly higher than startingmaterial. The reaction contents were poured into cold water andextracted with EtOAc. The organic layer was washed twice with saturatedcold sodium thiosulfate, saturated NaHCO₃, and with water. The productwas dried over anhydrous sodium sulfate and filtered to remove thedrying agent. The solvent was removed in vacuo which afforded a lightyellow waxy solid. Then, the product was dissolved in THF and thenconcentrated in vacuo twice at low temperatures to remove any residualsolvents not desired for the next step to afford 380 grams of2-Iodomethyl-3-(tri-O-benzyl-α-L-C-fucopyranoside)-1-propene (6) (97%).This material should be protected from heat and light, and usedimmediatly. A typical scale for this reaction is: 331 g startingmaterial, resulting in a yield of 380 g Product.

The product is depicted below: ##STR37##

EXAMPLE 7

2,3,4-Tri-O-benzyl-α-L-C-Fucopyranoside allylbromide reagent (7)

To a stirred suspension of 42.72 g LiBr (493 mmole, 5 mmole equiv.) in197 ml THF 50.0 g2-chloromethyl-3-(tri-O-benzyl-α-L-C-fucopyranoside)-1-propene (98.6mmole, 1 mmole equiv.) was added and the reaction was heated to refluxfor 3 hours, and then allowed to cool to room temperature. The reactionwas complete as assayed by TLC (product Rf slightly higher than startingmaterial). The TLC conditions used were 10% ethyl acetate in hexanes(v/v). The reaction contents were concentrated to half of the originalvolume of THF, poured into cold water and then extracted with EtOAc. Theorganic layer was washed twice with water, 1.0M HCl and again withwater. The product was dried over anhydrous sodium sulfate and filteredto remove the drying agent. The solvent was removed in vacuo whichafforded a light yellow solid. The product was dissolved in methanol andthen concentrated in vacuo at low temperatures twice to remove anyresidual solvents. The product was dissolved in 150 ml warm methanol andcooled to 0° C. overnight. Filtration of the solids results in 40.8grams as a white crystalline solid. Concentration of the mother liquorsto half of the original volume and again cooling to 0° C. overnight gavean additional 10.87 grams of a white crystalline solid. Combinedrecovery yielded 51.67 g of2-bromomethyl-3-(2,3,4-tri-O-benzyl-α-L-C-fucopyranoside)-1-propene;mp=51.5°-53° C., (95%).

The product gave the following analytical data:

¹ H-NMR (CDCl₃) δ 7.20-7.50 (m, 15H, aromatics), 5.2 (d, J=61.5 Hz, 2H,terminal vinyl), 4.50-4.90 (complex multiplet, 6H, benzylic, 4.25 (p,J=4.22 Hz, 1H, H-1), 4.04 (d, J=3.1 Hz, 2H, --CH₂ Br), 3.90 (m, 1H),3.75 (s, 1H), 2.50 (m, 2H), 1.25 (d, 3H). ¹³ C-NMR (CDCl₃) δ 1423.11alkene (e), 138.77 aromatic (e), 138.53 aromatic (e), 138.26 aromatic(e), 128.17 aromatic (e), 127.86 aromatic (o), 127.45 aromatic (o),127.34 aromatic (o), 117.00 alkene (e), 76.69 (o), 76.16 (o), 73.46 (e),73.11 (e), 69.9 (o), 68.46 (o), 37.03 --CH₂ Br (e), 30.54 allylic (e),15.61 fucose methyl (o). Analytical Calculated for C₃₁ H₃₅ BrO₄ : C,67.51; H, 6.40. Found: C, 67.81; H, 6.56.

In general, the reagent ratios for the remaining per-O-acetylatedcarbohydrates were for example:1,2,3,4,6-penta-O-acetyl-D-galactopyranoside (1.00 mmole equiv.) and2-chloromethyl-3-trimethylsilyl-1-propene (2.00 mmole equiv.) weredissolved in acetonitrile (1.3M). Boron trifluoride etherate (2.00 mmoleequiv.) and trimethylsilyltrifluoromethane sulfonate (0.400 mmoleequiv.) were carefully added neat at room temperature. The reaction wasrefluxed for 6 hours and worked up as described. TLC 30% ethyl acetatein hexane.

EXAMPLE 8

The Carbon Glycoside Formation with Acetyl Protected Sugar ##STR38##

2-Chloromethyl-3-(tri-O-acetyl-α-L-C-fucopyranoside)-1-propene (8)

50.0 g 1,2,3,4-tetra-O-acetyl-L-fucopyranose (5) (150.5 mmole, 1.00mmole equiv.) and 36.7 g (40.9 ml)2-chloromethyl-3-trimethylsilyl-1-propene (225.7 mmole, 2.00 mmoleequiv.) were dissolved in 116 ml acetonitrile; subsequently, 74.8 gboron trifluoride etherate (526.8 mmole, 3.50 mmole equiv.) and 13.4 g(11.6 ml) trimethylsilyltrifluoromethanesulfonate (60.2 mmoles, 0.40mmole equiv.) were carefully added after the addition of the Lewis acids(preferentially trimethyltrifluoromethanesulfonate and boron trifluorideetherate), the reaction was slowly warmed to reflux and maintained atreflux for 6 hours. The reaction was terminated by cooling to roomtemperature, pouring the reaction contents on 100 ml of water, followedby extraction of the crude α-C-glycoside with ethyl acetate. Theheterogenous layers were separated and the organic phase was washed withportions of water, saturated sodium bicarbonate, 1.0M hydrochloric acidand brine. The crude extract was dried over anhydrous sodium sulfate,filtered, and the solvent was removed in vacuo to afford an oil that waspurified by column chromatography on Baker grade flash silica gel (47-61mm) (ratio of 20 to 1), eluted with 10% ethyl acetate in hexanes.Concentration in vacuo afforded 46.4 g of2-chloromethyl-3-(2,3,4-tri-O-acetyl-α-L-C-fucopyranoside)-1-propene(85%, oil).

Under these reaction conditions, the1,2,3,4-tetra-O-acetyl-L-fucopyranoside reaction mixture turned a darkbrown to black color, and TLC monitoring showed the transformation fromstarting material to the (α-C-glycoside proceeded as expected. Twocompounds, distinguishable by TLC, were isolated by columnchromatography on silica gel by elution with 10% ethyl acetate inhexane. As determined by NMR analysis, the major compound was the2,3,4-tri-O-acetyl-α-C-L-fucopyranoside (85%), and a minor compound wasa small amount of starting material. The baseline material observed byTLC probably representing a third unidentified molecule.

The product gave the following analytical data:

Reaction yield: 85%, and the compound isolated as an oil. ¹ H-NMR(CDCl₃) δ, 5.3 (m, 1H), 5.2 (m, 2H), 5.2(s, 1H), 5.05 (s, 1H), 4.38 (m,J=3.48 Hz, 1H, H-1), 4.09 (s, 2H), 3.95 (dq, J=1.71 Hz and 4.70 Hz, 1H),2.6 (dd, J=11.39 Hz, 1H), 2.4 (dd, J=3.42 Hz, 1H), 2.15 (s, 3H), 2.05(s, 3H), 1.98 (s, 3H), 1.09 (d, J=6.41 Hz, 3H). ¹³ C-NMR (CDCl₃) δ171.03 acetyl (e), 170.66 acetyl (e), 170.38 acetyl (e), 142.06 alkene(e), 117.72 alkene (e), 71.66 ring (o), 71.19 ring (o), 68.94 ring (o),68.40 ring (o), 66.33 ring (o), 48.51 allylic (chloride side) (e), 29.50allylic (e), 20.77 (o), 20.71 (o), 20.64 (o), 16.53 L-fucose methylgroup (o). IR 2985, 1746, 1646 cm⁻¹. Mass Spec. (LSIMS with mNBA andNaOAc) 385.1 (MNa⁺), 363.2 (MH⁺). Analytical Calculated for C₁₆ H₂₃ ClO₇: C, 52.97; H, 6.39. Found: C, 52.66; H, 6.40.

EXAMPLE 9

Deprotection of Fucose:

The deprotection of the fucose reagent was done in methanol with acatalytic amount of sodium metal added to the stirring methanol. Thereaction was terminated by the careful addition of 1.0M HCl until the pHwas approximately 2. The solvent was removed in vacuo. The deprotectedfucose is reflected in the following structure: ##STR39##

The product gave the following analytical data:

The reaction was quantitative, mp=185°-186.5° C. ¹ H-NMR (CDCl₃) δ, 5.02(d, J=42.8, 2H, terminal vinyl), 4.01 allylic --CH₂ Cl (s, 2H), 3.89 (p,J=3.91 Hz, 1H, H-1), 3.69 (m, 2H, H-2 & 5), 3.45 (m, 2H, H-3 & 4), 2.36(m, 2H, allylic), 0.97 (d, J=6.47 Hz, 3H). ¹³ C-NMR (CD₃ OD) δ 145.35alkene (e), 117.18 alkene (e), 75.35 ring (o), 72.84 ring (o), 72.34ring (o), 69.88 ring (o), 69.15 (o), 49.34 --CH₂ Cl (e), 29.50 allylic(e), 17.05 L-fucose methyl (o). Mass Spec. (LSIMS with Gly) 237.1 (MH⁺).Analytical Calculated for C₁₀ H₁₇ ClO₄ : C, 50.74; H, 7.24. Found C,50.63; H, 7.43.

EXAMPLE 10

2-Chloromethyl-3-(tetra-O-acetyl-α-L-C-glucopyranoside)1-propene (10)

The reaction conditions used for the α-C-glycosidation of1,2,3,4-tetra-O-acetyl-L-fucopyranoside were applied to1,2,3,4,6-penta-O-acetyl-D-glycopyranose, and yielding the expectedα-C-glycosides of α-C-glucose (20%). NMR analysis of the α-C-glycosidecarbon shifts (CDCl₃) for the added C-3 unit in the acetyl protectedsugars showed a chemical shift around δ 48 for the --CH₂ Cl allyliccarbon and δ 28 for the allylic carbon which forms the C-glycoside atthe C-1 carbohydrate position, and the alkene shifts were around δ 142and δ 117. The carbon shifts for the allylic chloride side chain in thebenzylated sugars and in the acetyl protected sugars were comparable.The α-carbon glycoside derivative of glucose is shown below: ##STR40##

The product gave the following analytical data:

Reaction yield: 20%, and the compound isolated as an oil. ¹ H-NMR(CDCl₃) δ, 5.26 (t, J=9.10 Hz, 1H, H-3), 5.10 (d, J=45.12 Hz, 2H,terminal vinyl), 5.02 (m, 1H, H-3), 5.10 (d, J=45.12 Hz, 2H, terminalvinyl), 5.02 (m, 1H, H-2), 4.90 (t, J=8.97 Hz, 1H, H-4), 4.33 (m, 1H,H-1), 4.13 (dd, J=5.44 Hz, 1H, H-6), 3.98 (dd, J=2.62 Hz, 1H, H-6), 4.05(s, 2H, --CH₂ Cl), 3.86 (m, 1H, H-5), 2.61 (dd<J=11.54 Hz, 1H), 2.38(dd, J=3.17 Hz, 1H), 1.99 (s, 3H, acetyl), 1.98 (s, 3H, acetyl), 1.96(s, 3H, acetyl), 1.95 (s, 3H, acetyl). ¹³ C-NMR (CDCl₃) δ 172.03 acetyl(e), 171.54 acetyl (e), 171.04 acetyl (e), 170.99 acetyl (e), 142.33alkene (e), 118.96 alkene (e), 72.55 ring (o), 71.57 ring (o), 71.43ring (o) 70.49 ring (o), 70.13 ring (o), 63.63 C-6 ring (e), 49.29 --CH₂Cl (e), 30.15 allylic (e), 22.11 acetyl groups (o), 22.06 acetyl groups.IR 2958, 1729, 1646 cm⁻¹.

EXAMPLE 11

2-Chloromethyl-3-(tetra-O-acetyl-α-L--C-galactopyranoside)-1-propene(11)

The reaction conditions used for the α-C-glycosidation of1,2,3,4-tetra-O-acetyl-L-fucopyranoside were applied to the1,2,3,4,6-penta-O-acetyl-D-galactopyranose yielding the expectedα-C-glycosides of α-C-galactose (74%).1,2,3,4,6-penta-O-Acetyl-D-galactopyranoside (1.00 mmole equiv.) and2-chloromethyl-3-trimethylsilyl-1-propene (2.00 mmole equiv.) weredissolved in acetonitrile (1.3 m). Boron trifluoride etherate (2.00mmole equiv.) and trimethylsilyltrifluoromethane sulfonate (0.40 mmoleequiv.) were carefully added neat at room temperature. The reaction wasrefluxed for 6 hours and worked up as described; TLC: 30% ethyl acetatein hexanes.

NMR analysis of the α-C-glycoside carbon shifts (CDCl₃) for the addedC-3 unit in the acetyl protected sugars showed a chemical shift around δ48 for the --CH₂ Cl allylic carbon and δ 28 for the allylic carbon whichforms the C-glycoside at the C-1 carbohydrate position, and the alkeneshifts were around δ 142 and δ 117. The carbon shifts for the allylicchloride side chain in the benzylated sugars in the acetyl protectedsugars were comparable. The α-carbon glycoside derivative of galactoseis shown below: ##STR41##

The product gave the following analytical data:

Reaction yield: 74%, mp=80°-82° C. ¹ H-NMR (CDCl₃) δ, 5.31 (br, 1H),5.16 (m, 2H), 5.05 (d, J=47.17 Hz, 2H, terminal vinyl), 4.33 (m, J=3.54,1H, H-1), 4.1-3.9 (m, 3H), 4.02 (s, 2H), 2.52 (dd, J=11.41, 1H), 2.28(dd, J=2.75, 1H), 2.01 (s, 3H, acetyl), 1.98 (s, 3H, acetyl), 1.91 (s,6H, acetyl). ¹³ C-NMR (CDCl₃) δ 170.18 acetyl (e), 169.81 acetyl (e),169.97 acetyl (e), 169.53 acetyl (e), 141.04 alkene (e), 117.17 alkene(e), 70.64 ring (o), 68.09 ring (o), 67.79 ring (o), 67.55 ring (o),67.42 ring (o), 62.32 C-6 ring (e), 47.65 --CH2Cl (e), 28.86 allylic(e), 20.53 acetyl group (o), 20.47 acetyl group (o), 20.41 acetyl group(o). IR 2958, 1729, 1646 cm⁻¹. Mass Spec. (LSIMS with mNBA and NaOAc)443.1 (MNa⁺), 421.2 (MH⁺). Analytical Calculated for C₁₈ H₂₅ ClO₉ : C,51.37; H, 5.99. Found: C, 51.47; H, 6.15.

EXAMPLE 12

2-Chloromethyl-3-(tetra-O-acetyl-α-L-C-mannoopyranoside)-1-propene (12)

The reaction conditions used for the α-C-glycosidation of1,2,3,4-tetra-O-acetyl-L-fucopyranoside were applied to1,2,3,4,6-penta-O-acetyl-D-mannopyranose yielding the expectedα-C-glycoside of α-C-mannose (80%).

NMR analysis of the α-C-glycoside carbon shifts (CDCl₃) for the addedC-3 unit in the acetyl protected sugars showed a chemical shift around δ48 for the --CH₂ Cl allylic carbon and δ 28 for the allylic carbon whichforms the C-glycoside at the C-1 carbohydrate position, and the alkeneshifts were around δ 142 and δ 117. The carbon shifts for the allylicchloride side chain in the benzylated sugars and in the acetyl protectedsugars were comparable. The a-carbon glycoside derivated of mannose isshown in the following scheme: ##STR42##

The product gave the following analytical data:

Reaction yield: 80%, (compound isolated as an oil). ¹ H-NMR (CDCl₃) δ5.13 (m, 3H), 5.12 (d, J=41.76 Hz, 2H, terminal vinyl, 4.20 (q, J=6.41Hz, 1H, H-1), 4.05 (m, 2H), 4.04 (d, J=1.65 Hz, 2H, 3.85 (m, J=269 Hz,1H), 2.60 (dd, J=10.32 Hz, 1H), 2.39 (dd, J=4.52 Hz, 1H), 2.39 (dd,J=4.52 Hz, 1H), 2.03 (s, 3H, acetyl), 1.98 (s, 3H, acetyl), 1.93 (s, 3H,acetyl). ¹³ C-NMR (CDCl₃) δ 1.70.28 acetyl (e), 169.89 acetyl (e),169.66 acetyl (e), 169,37 acetyl (e), 140.43 alkene (e), 117.61 alkene(e), 73.06 ring (o), 70.52 ring (o), 70.07 ring (o), 68.47 ring (o),66.52 ring (o), 62.04 Ch₂ (e), 47.47 --CH₂ Cl (e), 31.95 allylic (e),20.67 acetyl CH₃ (o), 20.50 acetyl CH₃ (o), 20.47 acetyl CH₃ (o), 20.43acetyl CH₃ (o). IR 2958, 1729, 1646 cm⁻¹. Mass Spec. (LSIMS with mNBAand NaOAC) 443.0 (MNA⁺), 421.3 (MH⁺).

EXAMPLE 13

Preparation Of 1-Deoxy-1-α-(2-Carboxy-Naphthyl-6-Oxyethyl)-L-Fucose (13)

A solution of ethyl 6-hydroxyl-2-naphthate (49, 89 mg, 0.42 mmole)(6-hydroxy-2-naphthoic acid was obtained from TCI America and convertedto the ester using standard esterification methods) and iodide (2, 177mg, 0.42 mmole) in dimethylforamide (1 ml) was stirred with potassiumcarbonate (174 mg) for 14 hours. The reaction mixture was worked up withwater (30 ml) and ethyl acetate (100 ml). The organic layer was washedwith brine (30 ml), dried over sodium sulfate, filtered andconcentrated. The crude product was purified on a silica gel column(hexane ethyl acetate, 4:1) to provide the triacetate of 3 (120 mg,55%).

A solution of the triacetate in methanol (30 ml) was stirred with 1Normal sodium hydroxide (3 ml) for 4 hours. The mixture was acidifiedwith 6N hydrochloride and solid collected by filtration. This solid waswashed with water, ethyl acetate and suction dried to provide naphthoicacid (13) (24 mg).

EXAMPLE 14

Preparation Of 3,7-Di-(1-Deoxy-1-α-Fucosyl-Ethyleneoxy)-2-Naphthoic Acid(14)

A solution of ethyl 3,7-dihydroxy-2-naphthoate (62 mg, 0.26 mmole) (theacid was obtained from Aldrich and it was converted to the ester usingstandard techniques) 2 mg, and iodide 2 (242 mg) in dimethylforamide(0.5 ml) was heated at 55° C. with potassium carbonate (175 mg) for 4hours. An additional amount of the iodide 2 (80 mg) was added to themixture which was heated at 55° C. overnight. The mixture was worked upwith water (50 ml) and ethyl acetate (2×75 ml). The ethyl acetateextracts were washed with brine (30 ml), dried over sodium sulfate,filtered and concentrated. The crude product was purified on a silicagel column (hexane:ethyl acetate, 1.5:1) to provide the triacetate of 17(130 mg, 65% yield).

A solution of the triacetate (120 mg) in methanol (1 ml) was stirredwith 2N potassium hydroxide (1.1 ml) at room temperature overnight. Thiswas acidified with 6N hydrochloride solution and concentrated todryness. The residue was dissolved in methanol (10 ml) and solid removedby filtration. The filtrate was concentrated to provide naphthoic acid(14) (70 mg).

EXAMPLE 15

Preparation Of 3,5-Di-(1-Deoxy-1-α-Fucosyl-Ethyleneoxy)-2-Naphthoic Acid(15)

When ethyl 3,5-dihydtoxy-2-naphthoate (prepared by esterification of thecorresponding acid) is substituted for ethyl 3,7-dihydroxy-2-naphthoatein Example 14, the identical process afforded the naphthoic acid (15).

EXAMPLE 16

Preparation Of6-(1-Deoxy-1-α-Fucosyl-Ethyleneoxy)-α-Methyl-2-Naphthalene Acetic Acid(16)

6-Hydroxy-α-methyl-2-naphthaleneacetic acid methyl ester is preparedfrom 6-methoxy-α-methyl-2-naphthaleneacetic acid by first converting themethyl ether to a hydroxyl group utilizing refluxing 48% HBr/AcOH(Greene and Wuts) and then preparing the corresponding methyl esterutilizing diazomethane.

The iodide 2 (286.6 mg) and 6-hydroxy-α-methylnaphthaleneacetic acidmethyl ester (154.0 mg) were dissolved in dimethylformamide (3 ml) andpotassium carbonate (277,6 mg) was added. The reaction was stirred atroom temperature for 12 hours, after which it was partitioned betweenwater (10 ml) 15 and ethyl acetate (50 ml). The layers were separatedand the organics were washed with water (3×10 ml) and brine (10 ml). Theorganic layer was dried over magnesium sulfate, filtered andconcentrated to dryness. Purification of the residue on silica gel (25%ethyl acetate/hexane) gave the desired triacetate (225.8 mg, 64% yield).

The above triacetate product was dissolved in methanol (1.70 ml) and 2Nsodium hydroxide solution (1.70 ml) was added. The reaction was stirredat room temperature for 13 hours, after which it was acidified with 6N-hydrochloride. After concentrating to dryness, the residue was washedwith water and dried under vacuum to give the desired product 19 (167mg, 100% yield).

EXAMPLE 17

Preparation Of 6-Glyceroxy-α-Methyl-2-Naphthalene Acetic Acid (17)

6-Hydroxy-α-methyl-2-naphthaleneacetic acid methyl ester is preparedfrom 6-methoxy-α-methyl-2-naphthaleneacetic acid by first converting themethyl ether to a hydroxyl group utilizing refluxing 48% HBr/AcOH(Greene and Wuts) and then preparing the corresponding methyl esterutilizing diazomethane.

Glycidol (2.95 ml) (obtained from Aldrich) and6-hydroxy-α-methylnaphthaleneacetic acid methyl ester (1.02 g) weredissolved in dimethylformamide (10 ml). Potassium carbonate (3. g) wasadded and the reaction was stirred at room temperature for 3 days. Afterdiluting with ethyl acetate (100 ml), the reaction was washed with water(5×10 ml). The water layer was wahed with ethyl acetate (20 ml) and thecombined ethyl acetate layers were washed with brine (20 ml). Afterconcentrating, the residue was purified on silica gel (70% ethylacetate/hexane) to give the desired methyl ester glycidol adduct (251.5mg, 19% yield) and the glycidyl ester glycidol adduct (200.3 mg, 13%yield).

The glycidyl ester glycidol adduct was dissolved in methanol (0.6 ml)and 2N sodium hydroxide (0.6 ml) was added. The reaction was stirred attemperature for 23 hours and acidified with 6N hydrochloride. Afterconcentrating the mixture to dryness, the residue was washed with waterand dried under vacuum to give the desired product 17 (125.9 mg, 75%yield).

EXAMPLE 18

1-Deoxy-α-1-Acetylenyl-1,2,4-tri-O-Benzyl-1-Fucose (18)

A solution of 1-O-acetyl-2,3,4-tri-O-benzyl-L-fucose (10 g, 21 mmol) andbistrimethylsilylacetylene (7.16 g, 42 mmol) in dichloromethane (50 ml)was cooled to -20° C. under argon. A solution of tin tetrachloride indichloromethane (21 mmol, 1M) was added and the mixture was stirred at-20° C. for 1 hour. The reaction was quenched with saturated aqueoussodium bicarbonate (50 ml), extracted with ethyl acetate (3×50 ml) andthe combined organic layers were washed with saturated aqueous sodiumbicarbonate (2×10 ml) and brine (20 ml), dried (MgSO₄), filtered andconcentrated, the residue was purified on silica gel (30% ethylacetate/hexane) to give the silylacetylenyl fucose (8.1 g, 75% yield).Water (10 ml) followed by potassium flouride (9.16 g, 158 mmol) wasadded to a solution of compound 70 (8.1 g, 15.76 mmol) in DMF (30 ml).The mixture was stirred at room temperature (2 h), diluted with water(100 ml) and extracted with 30% ethyl acetate/hexane (3×50 ml). Thecombined organic layers were washed with brine (50 ml), dried (MgSO₄),filtered and concentrated to give compound 18 (6.97 g, 100% yield).

EXAMPLE 19

Preparation of1-Deoxy-1-α-(m-Carboxy-Phenylethyl)-L-Fucose-Phenylalanine Amide (19)

Carbonyl diimidazole (2.62 g, 16.14 mmol) was added to a solution ofm-iodobenzoic acid (4 g, 16.14 mmol) in tetrahydrofuran (100 ml), themixture was stirred at room temperature (45 min). Phenylalanine benzylester p-toluenesulfonate (6.90 g, 16.14 mmol) was added, the mixture wasstirred (18), and concentrated to dryness. The organic phase was washedwith 1N aq. HCL (3×20 ml), saturated aqueous sodium bicarbonate (2×20ml) and brine (20 ml), dried (MgSO₄) and concentrated to give the amide(4.79 g, 61% yield).

The amide (1.28 g, 2.63 mmol) and compound 18 (1.16 g, 2.63 mmol) weredissolved in DMF (10 ml). Triethylamine (0.73 ml, 5.26 mmol) was addedfollowed by tetrakistriphenyl-phosphine palladium (100 mg) and copper(1)iodide (40 mg). The reaction was stirred at room temperature (6 h),diluted with water (100 ml), and extracted with ethyl acetate (3×20 ml).The combined organic phases were washed with 1N aq. HCl (40 ml),saturated aqueous sodium bicarbonate (40 ml) and brine (40 ml), dried(MgSO₄)and concentrated. The residue was purified on silica gel (20%ethyl acetate/hexane) to give the coupled product (0.6 g, 28% yield).

The coupled product (0.6 g, 0.75 mmo) was dissolved in ethyl acetate (5ml). 10% Palladium/carbon (0.6 g) was suspended in methanol (40 m) andthe ethyl acetate solution was added via cannula. After stirring underhydrogen for 4 days, the mixture was filtered through Celite and thefiltrate was concentrated to dryness giving the title compound 22 (0.24g, 72% yield).

EXAMPLE 20 ##STR43##

2-Naphthol (6.20 g, 43.01 mmole) and tetra-O-acetyl 2-deoxyglucose(14.28 g, 43.01 mole) were dissolved in acetonitrile (100 mL) andborontrifluoride etherate (10.58 mL) was added. The reaction was stirredat room temperature for 24 hours after which it was quenched withsaturated aqueous sodium bicarbonate (100 mL). The resulting mixture wasdiluted with ethyl acetate (200 mL) and washed with saturated aqueoussodium bicarbonate (2×50 mL) and brine (50 mL). After drying overanhydrous magnesium sulfate, the solution was filtered, concentrated,and purified on silica gel (30% ethyl acetate/hexane) to give thedesired product (10.54 g, 59%).

EXAMPLE 21 ##STR44##

2-Naphthol (8.72 g, 60.48 mmole) and tetra-O-acetyl 2-deoxygalactose(20.08 g, 60.48 mmole) were dissolved in acetonitrile (100 mL) andborontrifluoride etherate (14.88 mL) was added. The reaction and workupwere identical to that in example 20 giving the desired product (16.89g, 67%).

EXAMPLE 22 ##STR45##

2-Naphthol (1.00 g, 7.01 mmole) and tri-O-benzyl 1-O-acetylfucose (2.22g, 4.67 mmoles) were dissolved in acetonitrile (15 mL) and 4 Å molecularsieves (2.00 g) were added. After adding borontrifluoride etherate(1.72) mL), the reaction was stirred at room temperature for three daysand quenched with 6N HCl (10 mL). After stirring at room temperature for2 hours, the reaction was neutralized with saturated aqueous sodiumbicarbonate. The resulting mixture was washed with ethyl acetate (2×50mL) and brine (2×25 mL). After drying over anhydrous magnesium sulfate,the solution was filtered, concentrated and the product purified onsilica gel (10% ethyl acetate/hexane) to give the desired product (2.05g, 78%).

EXAMPLE 23 ##STR46##

6-O-Demethyl naproxen methyl ester (1.00 g, 4.35 mmole) and tri-O-benzyl1-O-acetylfucose (1.38 g, 2.90 mmoles) were dissolved in acetonitrile(10 mL) and 4 Å molecular sieves (2.00 g) were added. After addingborontrifluoride etherate (1.07 mL), the reaction was stirred at roomtemperature for two days and quenched with 6N HCl (10 mL). Afterstirring at room temperature for 40 minutes, the reaction wasneutralized with saturated aqueous sodium bicarbonate. The resultingmixture was washed with ethyl acetate (3×20 mL). After drying overanhydrous magnesium sulfate, the solution was filtered, concentrated andthe product purified on silica gel (hexane then 10% acetone/hexane then15% acetone/hexane) to give the desired product (1.44 g, 77%).

EXAMPLE 24 ##STR47##

6-O-Demethyl naproxen methyl ester and tetra-O-acetyl 2-deoxygalactoseare dissolved in acetonitrile and borontrifluoride etherate is added.The reaction and workup are identical to that in Example 20.

EXAMPLE 25 ##STR48##

The product from example 24 and1-deoxy-1-α-3-chloromethallyl)-2,3,4-tri-O-acetylfucose are dissolved indimethylformamide and potassium carbonate is added followed bytetrabutyl ammoniumiodide. After stirring at room temperature for 2days, the reaction is diluted with ethyl acetate and washed withsaturated aqueous sodium bicarbonate (3×) and brine (2×). The mixture isdried over anhydrous magnesium sulfate, filtered and concentrated. Theresidue is purified on silica gel to give the desired product.

EXAMPLE 26 ##STR49##

The product from Example 25 is dissolved in 1,2-dichlorobenzene andheated to 190 degrees C for six hours. After cooling to roomtemperature, reaction mixture is separated on silica gel giving thedesired product.

EXAMPLE 26 ##STR50##

Sodium metal (4 spheres) are washed in hexane and added to anhydrousmethanol (20 mL). 0.10 mL of the resulting sodium methoxide solution isadded to a solution of the product from Example 25 in anhydrous methanol(10 mL). After stirring at room temperature for 24 hours, the reactionis concentrated to dryness giving the desired product.

EXAMPLE 27 ##STR51##

The product from example 26 is dissolved in methanol and an equivalentvolume of aqueous 2N NaOH (2 equivalents) is added. The reaction isstirred at room temperature for 24 hours and neutralized methanol washedAmberlyst acidic ion exchange resin. Filtration and concentration of theresulting solution provides the desired product.

EXAMPLE 28 ##STR52##

The product from Example 26 is dissolved in methylene chloride/dimethylformamide (20/1) and TBDMS-CI (0.53 g, 3.51 mmoles) is added followed byimidazole (0.34 g, 5.02 mmoles). After stirring at room temperature for20 hours, TBDMS-Cl (0.53 g, 3.51 mmoles) and imidazole (0.34 g, 5.02mmoles) are added. Stirring is continued for an additional 20 hoursafter which, the reaction is quenched with methanol (10 mL). Afterstirring for 30 minutes at room temperature, the reaction isconcentrated to dryness and the product is purified on silica gel.

EXAMPLE 29 ##STR53##

The product from Example 28 is dissolved in pyridine and an equivalentvolume of acetic anhydride is added. After stirring at room temperaturefor three days, the reaction is concentrated to dryness and the residueis dissolved in ethyl acetate. After washing with 1N HCl (3×), saturatedaqueous sodium bicarbonate (6×), water, saturated aqueous copper sulfate(2×), and brine (10 mL), the organic phase is dried over anhydrousmagnesium sulfate, filtered and concentrated to dryness giving thedesired product (1.98 g, 81%).

EXAMPLE 30 ##STR54##

The product described in Example 29 is dissolved in tetrahydrofuran andtetrabutyl ammonium fluoride (1M in THF) is added. After stirring atroom temperature for five days, the reaction is concentrated.Purification of the residue on silica gel gives the desired product.

EXAMPLE 31 ##STR55##

The product described in Example 30 and methyl bromoacetate aredissolved in tetrahydrofuran and sodium hydride (60% in mineral oil) isadded. After stirring at room temperature for 2 days, the reaction isquenched with saturated aqueous sodium bicarbonate. The reaction isdiluted with ethyl acetate and washed with brine (2×). After drying overanhydrous magnesium sulfate, the organic phase is filtered andconcentrated. Purification of the residue on silica gel gives theproduct.

EXAMPLE 32 ##STR56##

Sodium metal (4 spheres) are washed in hexane and added to anhydrousmethanol (20 mL). 0.10 mL of the resulting sodium methoxide solution isadded to a solution of the product from Example 31 in anhydrousmethanol. After stirring at room temperature for 24 hours, the reactionis concentrated to dryness giving the desired product.

EXAMPLE 33 ##STR57##

The product described in Example 32 is dissolved in methanol and anequivalent volume of 2N aqueous sodium hydroxide (4 equivalents) isadded. The reaction is stirred at room temperature for 20 hours andneutralized with methanol washed Amberlyst acidic ion exchange resin.Filtration and concentration of the resulting solution provides thedesired product.

EXAMPLE 34 ##STR58##

Sodium metal (4 spheres) were washed in hexane and added to anhydrousmethanol (20 mL). 0.20 mL of the resulting sodium methoxide solution isadded to a solution of the product from Example 28 in anhydrousmethanol. After stirring at room temperature for 24 hours, the reactionis neutralized with amberlite acidic ion exchange resin. After removalof the resin by filtration, the filtrate is concentrated to drynessgiving the desired product.

EXAMPLE 35 ##STR59##

The product from Example 34 is dissolved in 2,2-dimethoxypropane andcamphorsulfonic acid is added. After stirring at room temperature for 24hours, the reaction is concentrated to dryness. The residue is dissolvedin ethyl acetate and washed with saturated aqueous sodium bicarbonate(3×) and brine. After drying over anhydrous magnesium sulfate, theorganic phase is filtered and concentrated to dryness giving the desiredproduct.

EXAMPLE 36 ##STR60##

The product described in Example 35 and1-deoxy-1-α-(3-chloromethallyl)-2,3,4-tri-O-acetylfucose are dissolvedin dimethylformamide and potassium carbonate is added followed bytetrabutyl ammoniumiodide. After stirring at room temperature for 6days, the reaction is diluted with ethyl acetate and washed withsaturated aqueous sodium bicarbonate (3×) and brine (2×). The mixture isdried over anhydrous magnesium sulfate, filtered and concentrated. Theresidue is purified on silica gel to give the desired product.

EXAMPLE 37 ##STR61##

This product is prepared from the product of Example 36 usingmethodology similar to that used in Example 34.

EXAMPLE 38 ##STR62##

This product is prepared from the product of Example 37 usingmethodology similar to that used in Example 34.

EXAMPLE 39 ##STR63##

This product is prepared from the product of example 38 usingmethodology similar to that used in Example 33.

EXAMPLE 40 ##STR64##

The product from Example 39 is dissolved in tetrahydrofuran and anequivalent volume of 6N hydrochloric acid is added. After stirring atroom temperature for 24 hours, the reaction is concentrated to drynessto give the desired product.

EXAMPLE 41 ##STR65##

This product is prepared from the product of Example 40 usingmethodology similar to that used in Example 26.

EXAMPLE 42 ##STR66##

The product from example 20 (10.54 g, 25.34 mmoles) and1-deoxy-1-α-(3-chloromethallyl)-2,3,4-tri-O-acetylfucose (10.09 g, 27.87mmoles) were dissolved in dimethylformamide (100 mL) and potassiumcarbonate (7.00 g, 50.67 mmoles) was added followed by tetrabutylammoniumiodide (0.94 g, 2.53 mmoles). After stirring at room temperaturefor 2 days, the reaction was diluted with ethyl acetate (300 mL) andwashed with saturated aqueous sodium bicarbonate (3×50 mL) and brine(2×50 mL). The mixture was dried over anhydrous magnesium sulfate,filtered and concentrated. The residue was purified on silica gel (30%ethyl acetate/hexane) to give the desired product (12.83 g, 68%).

EXAMPLE 43 ##STR67##

The product from Example 21 (16.89 g, 40.60 mmoles) and1-deoxy-1-α-(3-chloromethallyl)-2,3,4-tri-O-acetylfucose (15.43 g, 42.63mmoles) were dissolved in dimethylformamide (100 mL) and potassiumcarbonate (11.22 g, 81.20 mmoles) was added followed by tetrabutylammoniumiodide (1.50 g, 4.06 mmoles). After stirring at room temperaturefor 2 days, the reaction was diluted with ethyl acetate (300 mL) andwashed with saturated aqueous sodium bicarbonate (3×50 mL)and brine(2×50 mL). The mixture was dried over anhydrous magnesium sulfate,filtered and concentrated. The residue was purified on silica gel (30%ethyl acetate/hexane) to give the desired product (27.12 g, 90%).

EXAMPLE 44 ##STR68##

The product from Example 42 (6.13 g, 8.26 mmoles) was dissolved in1,2-dichlorobenzene (16.60 mL) and heated to 190 degrees C for sixhours. After cooling to room temperature, reaction mixture was separatedon silica gel giving the desired product (3.23 g, 53%).

EXAMPLE 45 ##STR69##

The product from Example 43 (6.38 g, 8.60 mmoles) was dissolved in1,2-dichlorobenzene (17.00 mL) and heated to 190 degrees C for sixhours. After cooling to room temperature, reaction mixture was separatedon silica gel giving the desired product (2.28 g, 36%).

EXAMPLE 46 ##STR70##

Sodium metal (4 spheres) were washed in hexane and added to anhydrousmethanol (20 mL). 0.10 mL of the resulting sodium methoxide solution wasadded to a solution of the product from Example 42 (3.05 g, 4.11 mmoles)in anhydrous methanol (20 mL). After stirring at room temperature for 24hours, the reaction was concentrated to dryness giving the desiredproduct (2.10 g, 100%).

EXAMPLE 47 ##STR71##

Sodium metal (4 spheres) were washed in hexane and added to anhydrousmethanol (20 mL). 0.10 mL of the resulting sodium methoxide solution wasadded to a solution of the product from Example 43 (4.97 g, 6.70 mmoles)in anhydrous methanol (20 mL). After stirring at room temperature for 24hours, the reaction was concentrated to dryness giving the desiredproduct (3.45 g, 100%).

EXAMPLE 48 ##STR72##

Sodium metal (4 spheres) were washed in hexane and added to anhydrousmethanol (20 mL). 0.10 mL of the resulting sodium methoxide solution wasadded to a solution of the product from Example 44 (3.23 g, 4.35 mmoles)in anhydrous methanol (10 mL). After stirring at room temperature for 24hours, the reaction was concentrated to dryness giving the desiredproduct (2.14 g, 100%).

EXAMPLE 49 ##STR73##

Sodium metal (4 spheres) were washed in hexane and added to anhydrousmethanol (20 mL). 0.10 mL of the resulting sodium methoxide solution wasadded to a solution of the product from Example 45 (2.28 g, 3.07 mmoles)in anhydrous methanol (10 mL). After stirring at room temperature for 24hours, the reaction was concentrated to dryness giving the desiredproduct (1.55 g, 100%).

EXAMPLE 50 ##STR74##

The product from Example 46 (1.64 g, 3.35 mmoles) was dissolved inmethylene chloride (20 mL) and dimethyl formamide (1 mL) and TBDMS-Cl(0.53 g, 3.51 mmoles) was added followed by imidazole (0.34 g, 5.02mmoles). After stirring at room temperature for 20 hours, TBDMS-Cl (0.53g, 3.51 mmoles) and imidazole (0.34 g, 5.02 mmoles) were added. Stirringwas continued for an additional 20 hours after which, the reaction wasquenched with methanol (10 mL). After stirring for 30 minutes at roomtemperature, the reaction was concentrated to dryness and the productwas purified on silica gel (10% methanol/chloroform, 1.81 g, 90%).

EXAMPLE 51 ##STR75##

This product (1.65 g, 46%) was prepared from the compound described inExample 47 using similar conditions to those described in Example 50.

EXAMPLE 52 ##STR76##

This product (1.76 g, 80%) was prepared from the compound described inExample 48 using similar conditions to those described in Example 50.

EXAMPLE 53 ##STR77##

This product (1.02 g, 64%) was prepared from the compound described inExample 49 using similar conditions to those described in Example 50.

EXAMPLE 54 ##STR78##

The product from Example 50 (1.81 g, 3.00 mmoles) was dissolved inpyridine (5.00 mL) and acetic anhydride (5.00 mL) was added. Afterstirring at room temperature for three days, the reaction wasconcentrated to dryness and the residue was dissolved in ethyl acetate(50 mL). After washing with 1N HCl (3×10 mL), saturated aqueous sodiumbicarbonate (6×10 mL), water (10 mL), saturated aqueous copper sulfate(2×10 mL), and brine (10 mL), the organic phase was dried over anhydrousmagnesium sulfate, filtered and concentrated to dryness giving thedesired product (1.98 g, 81%).

EXAMPLE 55 ##STR79##

This product (3.04 g, 70%) was prepared from the compound described inExample 51 using similar conditions to those described in Example 54.

EXAMPLE 56 ##STR80##

This product (2.07 g, 83%) was prepared from the compound described inExample 52 using similar conditions to those described in Example 54.

EXAMPLE 57 ##STR81##

This product (1.24 g, 86%) was prepared from the compound described inExample 53 using similar conditions to those described in Example 54.

EXAMPLE 58 ##STR82##

The product described in Example 54 (1.98 g, 2.43 mmoles) was dissolvedin tetrahydrofuran (10 mL) and tetrabutyl ammoniumfluoride (1M in THF,2.68 mL, 2.68 mmoles) was added. After stirring at room temperature forfive days, the reaction was concentrated. Purification of the residue onsilica gel (50% ethyl acetate/hexane) gave the desired product (1.29 g,76%).

EXAMPLE 59 ##STR83##

This product (0.87 g, 69%) was prepared from the compound described inExample 55 using similar conditions to those described in Example 58.

EXAMPLE 60 ##STR84##

This product (0.96 g, 63%) was prepared from the compounds described inExample 56 using similar conditions to those described in Example 58.

EXAMPLE 61 ##STR85##

This product (0.83 g, 94%) was prepared from the compound described inExample 57 using similar conditions to those described in Example 58.

EXAMPLE 62 ##STR86##

The compound described in Example 58 (1.29 g, 1.84 mmoles) was dissolvedin carbon tetrachloride (4 mL), acetonitrile (4 mL), and water (6 mL).After adding sodium periodate (1.58 g, 7.37 mmoles), the reaction wascooled to zero degrees C. Ruthenium trichloride hydrate (8.4 mg, 0.41mmoles) was added and the reaction was vigorously stirred for four hourswhile warming to room temperature. The reaction was then diluted withethyl acetate (100 mL) and washed with water (2×20 mL), saturatedaqueous sodium bicarbonate (3×20 mL), and brine (20 mL). After dryingover anhydrous magnesium sulfate, organic phase was filtered,concentrated, and purified on silica gel (70% ethyl acetate/hexane)giving the product (0.86 g, 65%).

EXAMPLE 63 ##STR87##

The product described in Example 62 (0.86 g, 120 mmoles) was dissolvedin methanol (7.23 mL) and 2N aqueous sodium hydroxide (7.23 mL) wasadded. After stirring at room temperature for 24 hours, the methanol wasevaporated and the aqueous residue was acidified with 6N HCl to pH=2.The water was then removed on a lyophilizer. The salt components of theresidue were removed by washing with minimal amounts of methanol andremoving the solids by filtration. Concentration of the filtrateprovided the product (0.62 g, 100%).

EXAMPLE 64 ##STR88##

The product described in Example 60 (0.96 g, 1.29 mmoles) and methylbromoacetate (0.18 mL, 1.94 mmoles) were dissolved in tetrahydrofuran(2.00 mL) and sodium hydride (60% in mineral oil, 62 mg, 1.55 mmoles)was added. After stirring at room temperature for 2 days, the reactionwas quenched with saturated aqueous sodium bicarbonate (10 mL). Thereaction was diluted with ethyl acetate (50 mL) and washed with brine(2×25 mL). After drying over anhydrous magnesium sulfate, the organicphase was filtered and concentrated. Purification of the residue onsilica gel (50% ethyl acetate/hexane) gave the product (0.53 g, 50%).

EXAMPLE 65 ##STR89##

Sodium metal (4 spheres) were washed in hexane and added to anhydrousmethanol (20 mL). 0.10 mL of the resulting sodium methoxide solution wasadded to a solution of the product from Example 64 (0.53 g, 0.65 mmoles)in anhydrousmethanol (10 mL). After stirring at room temperature for 24hours, the reaction was concentrated to dryness giving the desiredproduct (0.36 g, 99%).

EXAMPLE 66 ##STR90##

The product described in Example 65 (0.36 g, 0.64 mmoles) was dissolvedin methanol (0.64 mL) and 2N aqueous sodium hydroxide (0.64 mL, 1.28mmoles) was added. The reaction was stirred at room temperature for 20hours and the methanol was removed under reduced pressure. The residuewas dissolved in water (20 mL) and the water was removed on alyophilizer giving the desired product (0.39 g, 100%).

EXAMPLE 67 ##STR91##

Sodium metal (4 spheres) were washed in hexane and added to anhydrousmethanol (20 mL). 0.20 mL of the resulting sodium methoxide solution wasadded to a solution of the product from Example 20 (12.29 g) inanhydrous methanol (20 mL). After stirring at room temperature for 24hours, the reaction was neutralized with amberlite acidic ion exchangeresin. After removal of the resin by filtration, the filtrate wasconcentrated to dryness giving the desired product (7.90 g, 92%).

EXAMPLE 68 ##STR92##

The product from Example 67 (7.90 g, 27.24 mmoles) was dissolved in2,2-dimethoxypropane (100 mL) and camphorsulfonic acid (0.32 g, 1.36mmoles) was added. After stirring at room temperature for 24 hours, thereaction was concentrated to dryness. The residue was dissolved in ethylacetate (300 mL) and washed with saturated aqueous sodium bicarbonate(3×50 mL) and brine (50 mL). After drying over anhydrous magnesiumsulfate, the organic phase was filtered and concentrated to drynessgiving the desired product (8.44 g, 94%).

EXAMPLE 69 ##STR93##

The product described in Example 68 (8.44 g, 25.58 mmoles) and1-deoxy-1-α-(3-chloromethallyl)-2,3,4-tri-O-acetylfucose (10.18 g, 28.13mmoles) were dissolved in dimethylformamide (100 mL) and potassiumcarbonate (7.07 g, 51.15 mmoles) was added followed by tetrabutylammoniumiodide (0.94 g, 2.53 mmoles). After stirring at room temperaturefor 6 days, the reaction was diluted with ethyl acetate (300 mL) andwashed with saturated aqueous sodium bicarbonate (3×50 mL) and brine(2×50 mL). The mixture was dried over anhydrous magnesium sulfate,filtered and concentrated. The residue was purified on silica gel (50%.ethyl acetate/hexane) to give the desired product (14.04 g, 84%).

Example A Selectin Binding

An ELISA assay was employed that uses recombinant fusion proteinscomposed of extracellular portions of the human selectins joined tohuman immunoglobulin heavy chain CH₃, CH₂, and hinge regions. See, forexample, Walz et al., Science (1990) 250:1132; Aruffo et al., Cell(1991) 67:35; Aruffo et al., Proc. Natl. Acad. Sci. USA (1992) 89:2292.The assay is well known in the art, and generally consists of thefollowing three steps:

I. 2,3 sLe^(x) glycolipid (25 picomole/well) was transferred intomicroliter well as solutions and then evaporated off. Excess, whichremained unattached, was washed off with water. The wells were thenblocked with 5% BSA at room temperature for an hour and then washed withPBS containing 1 mM calcium.

II. Preparation of "multivalent" receptor of the Selectin-IgG chimerawas carried out by combining the respective chimera 1 μg/ml) with biotinlabelled goat F(ab')2 anti-human IgG (Fc specific) andstreptavidin-alkaline phosphatase diluted 1:1000 in 1% BSPBS (1 mMcalcium) and incubating at 37° C. for 15 min. This allowed the solublemultivalent receptor complex to form.

III. Potential inhibitors such as compounds of formula I were allowed toreact with the soluble receptor at 37° C. for 45 min. This test assumesthat optimal binding, between the soluble phase receptor complex and theinhibitor (non-natural ligand), would have occurred within this timeframe. This solution was then placed in the microliter wells that wereprepared in step I. The plate was incubated at 37° C. for 45 minutes toallow the soluble receptor to bind to its natural ligand. In thepresence of a strong inhibitor only a few receptors should be free tobind to the microliter plate coated with the natural ligand.

The positive control is the signal produced by the soluble receptor whenit is allowed to react with 2,3 sLe^(x) glycolipid in the microliterwells in the absence of any inhibitor. This was considered 100% binding.The signal produced by the receptor that had been previously treatedwith an inhibitor (recorded as O.D.), was divided by the signal producedby the positive control and multiplied by 100 to calculate the %receptor bound to the well, or the percent of control binding. Severalof the compounds described herein were tested using this assay. Tables 2and 3 list the extent to which the invention compounds inhibit bindingof E, L and P-selectin to 2,3 sLe^(x) glycolipid in terms of IC₅₀values.

                                      TABLE 2                                     __________________________________________________________________________    Naphthoic Acid C-Glycosides                                                    ##STR94##                                                                                         E      L      P                                          R.sub.2                                                                              R.sub.3                                                                          R.sub.5                                                                          R.sub.6                                                                           R.sub.7                                                                           (IC.sub.50, mM)                                                                      (IC.sub.50, mM)                                                                      (IC.sub.50, mM)                            __________________________________________________________________________    CO.sub.2 H                                                                           H  H  OF.sub.2.sup.A                                                                    H   <0.5   <1.0   <0.5                                                            >1.5   >1.5   >1.5                                       CH(CH.sub.3)CO.sub.2 H                                                               H  H  OF.sub.2.sup.A                                                                    H   >4.0   >2.0   >2.0                                       CO.sub.2 H                                                                           OF.sub.2.sup.A                                                                   H  H   OF.sub.2.sup.A                                                                    >4.0   <2.0   <0.5                                                            >1.5   >1.5   >1.5                                       CO.sub.2 H                                                                           OF.sub.2.sup.A                                                                   OF.sub.2.sup.A                                                                   H   H   >4.0   <4.0   <2.0                                       CO.sub.2 H                                                                           H  H  O-DF.sub.2.sup.A                                                                  H   >1.5   >1.5   >1.5                                       CO.sub.2 H                                                                           OF.sub.2.sup.A                                                                   H  H   O-DF.sub.2.sup.A                                                                  >1.5   >1.5   >1.5                                       CH(CH.sub.3)CO.sub.2 H                                                               H  H  OF.sub.1.sup.A                                                                    H   >1.0   >1.0   >1.0                                       __________________________________________________________________________

                                      TABLE 3                                     __________________________________________________________________________    Naphthol-C-Glycosides                                                          ##STR95##                                                                                                           E       L      P                       EXAMPLE                                                                              R"      R.sub.5       R.sub.6                                                                            R.sub.7                                                                            (IC.sub.50, mM)                                                                       (IC.sub.50,                                                                          (lC.sub.50,             __________________________________________________________________________                                                          mM)                            H                                                                                      ##STR96##    H    F.sub.1.sup.A                                                                      Ins     Ins    Ins                            H                                                                                      ##STR97##    F.sub.1.sup.A                                                                      H    Ins     Ins    lns                            H                                                                                      ##STR98##    F.sub.1.sup.A                                                                      H    >4.0    >4.0   >4.0                           H                                                                                      ##STR99##    H    F.sub.1.sup.A                                                                      >1.5    >1.5   >1.5                           H                                                                                      ##STR100##   F.sub.1.sup.C                                                                      H    >1.5    >1.5   >1.5                           CH.sub.2 CO.sub.2 OH                                                                   ##STR101##   H    F.sub.1.sup.A                                                                      >2.0    >2.0   >2.0                    __________________________________________________________________________

In addition to the ligands described above, other ligands could beobtained by selecting more rigid spacers in order to maintain theappropriate statistical average distance between the sialic acid andfucose moieties in space thereby improving the inhibitory property ofsuch structures towards the selecting. Further modifications of thesecompounds e.g., attaching them through chemical linkages on appropriatemolecular supports and use of analogs or derivatives of sialic acid andL-fucose are also considered to be within the scope of the presentinvention.

Example B Flow Cytometric Assay for P-selectin Ligand

The interaction of P-selectin and its cellular ligand was studied usinga flow cytometric assay (Erbe, D. V. et al., J. Cell Biol. (1993)120:1227). HL60 cells (maintained in high glucose DME plus 10% HycloneFBS) were used in this assay. Before staining with P-selectin-IgG thecells were preincubated in Dulbecco's PBS/1% BSA/0.1% sodium azide/1%normal rabbit serum (staining medium) for 30-60 mins on ice. After thisinitial incubation, 1 μg of P-selectin-IgG was added to 100 μl aliquotsof 106 cells and incubated for 30-60 mins on ice. The cells were thenwashed with staining medium and resuspended in 100 μl of staining mediumto which was added 2 μl of a phycoerythrin-conjugated F(ab')² goatanti-human IgG (Fc specific). The cells were incubated for 15-30 mins onice, washed twice with staining medium, and resuspended in 0.5 ml ofstaining medium before flow cytometric analysis on a FACScan (BectonDickinson & Co., Mountain View, Calif.). To determine that the stainingwas an interaction of P-selectin with its ligand, the staining was alsodone in the presence of 10 mM EGTA. To determine the proteasesensitivity and the requirement for sialic acid of this interaction,HL-60 cells in D-PBS and 1% BSA were incubated with either trypsin orArthrobacter or Clostridium sialidases at 37° C. before resuspending instaining medium. To examine the ability of various carbohydrates toinhibit staining, 50 μg/ml fucoidin (Sigma Immunochemicals, St. Louis,Mo.), 50 μg/ml dextran sulfate (Pharmacia Fine Chemicals, Piscataway,N.J.), 10 mg/ml mannose-1-phosphate (Sigma Immunochemicals), or 10 mg/mlmannose-6-phosphate (Sigma Immunochemicals) was added to cellsimmediately before the addition of the P-selectin chimera. Eachcarbohydrate was then present until the cells were washed before theaddition of the second stage antibody. A potential complication of thisFACS assay arose from the use of selectin-IgG chimeras to stain cellswhich bear human IgG Fc receptors (FcΥR; Fanger, M. W., et al., Immunol.(1989) 10:92). Adding rabbit IgG (in the form of normal rabbit serum) tothe assay medium blocked this binding in most cases. Table 4 shows theresults (in terms of % inhibition) of the ability of compounds C and D,6-O-sulfo-hexanyl-α-L-furopyranoside, to inhibit P-selectin-IgG bindingto HL-60 cells lines. The structure of compound C is shown below:

                  TABLE 4                                                         ______________________________________                                         ##STR102##                                                                   Compound #  Concentration (mg/ml)                                                                       Inhibition (%)                                      ______________________________________                                        C           10            60                                                              50            80                                                              100           80                                                  D           10            50                                                              100           50                                                              200           60                                                  ______________________________________                                    

Example C Treatment of Sepsis

A number of the complications associated with sepsis arise from unwantedneutrophil extravasation and adhesion of the neutrophils to theendothelium. The invention compounds would be used to prevent or treatsepsis.

The effectiveness of these compounds would be shown in a baboon sepsismodel system as described by Taylor et al., J. of Clinical Inv., (1987),79:918, and by Taylor, et al., Circulatory Shock, (1988), 26:227.Briefly this model would consist of determining if the compounds areeffective in treating sepsis by preventing the death, or prolonging thelives of septic animals to either a letal or sublethal dose of E. coli.A lethal or sublethal dose of E. coli. consists of approximately 4×10¹⁰and 0.4×10¹⁰ organisms, respectively. Baboons that receive a lethal doseof E. coli invariably die within 16-32 hours. Taylor, et al., J. ofClinical Inv. (1987), 79:918, and Taylor et al., Circulatory Shock,(1988), 26:227.

Thus, the procedure would consist of using two administration routinesfor each of the compounds tested wherein they are delivered inphysiological saline. In the first, between 1 and 10 mg of compound perkg of body weight is administered in three separate doses at 24, 22, and21 hours before a lethal challenge of bacteria. Alternatively, compoundcan be administered in a single dose simultaneously with the bacterialchallenge. In both instances the compounds would considerably extend thelifetime of the baboons that receive the multiple or single dosetreatment and they would survive well beyond 48 hours.

Example D Reperfusion Injury Assay

Experiments were done to determine the effectiveness of compound C indecreasing adhesion of human neutrophils in the rabbit isolated heart.Addition of the human plasma to the rabbit isolated heart results inactivation of the complement components found within the plasma, whichin turn promotes an increase in the neutrophil accumulation. This modelis used to determine the effect of sLe^(x) analogues on inhibitingcomplement-induced neutrophil adhesion.

Hearts from New Zealand White rabbits were excised, mounted on amodified Langendorff apparatus and perfused with Krebs-Heinseleitbuffer. Cardiac functional parameters were monitored upon a Grass Model79D polygraph machine. 4% normal human plasma (NHP) was added to therecirculating buffer. Ten minutes after the addition of the plasma,Compound C (0.1 20 mg/ml) was added to the perfusate. After 15 minutesof perfusion with the plasma, 51-chromium labelled human neutrophils(1×10⁵ /ml) were added to the perfusate and allowed to recirculate foran additional 15 minutes. At the end of this time the hearts were washedwith fresh buffer to remove non-specifically bound neutrophils, driedand counted in a well-type gamma-counter. A concentration response curvewas generated using concentrations of 0.001, 0.01 and 0.1 mg/ml. Sixhearts were used for each of these concentrations. 30 Percent inhibitionof neutrophil accumulation was found. These results are expressed as thenumber of radiolabelled human neutrophils/mg of dry weight of the heart.

It should also be noted that the greatest degree of inhibition seenusing pharmacological agents, including a number of peptides derivedform P-selectin and antibodies directed against P-selectin and the CD11b/CD18 complex (Ma, Xin-liang, et al., Circulation (1993) 88-2:649),has been 40%. Compound C provides a degree of inhibition (30%) similarto any of the pharmacological agents tested thus far.

Based on the above results, it is apparent that the compounds of theinvention are useful for treating diseases, preferably diseases thathave an inflammatory component-Adult Respiratory Distress Syndrome(ARDS), ischemia and reperfusion injury, including strokes, mesentericand peripheral disease, organ transplantation, and circulatory shock (inthis case one or many organs might be damage following restoration ofblood flow).

What is claimed:
 1. A compound of the formula I: ##STR103## wherein R¹,R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ are independently selected from the groupconsisting of(a) --H, Y--B, alkyl of 1 to 4 carbon atoms optionallysubstituted with 1 to 2 lower alkyl groups,--W((CH₂)_(n) --B)_(t),--W((CH₂)_(m) --(CHR⁹)_(q) --(CH₂)_(m) --A)_(t) ; --OH, lower alkoxy,lower aryloxy, lower aralkoxy, lower alkoxyaryl, amino, --W((CH₂)_(n)--A)_(t), --O--CH₂ --C.tbd.C--B, --N(Ac)--CH₂ --C.tbd.C--B, --NH--CH₂--C.tbd.C--B, --N(CH₂ --C.tbd.C--B)₂, --N(Ac)CH₂ Ar--B, --NHCH₂ Ar--B,--N(CH₂ Ar--B)₂, --OCH₂ Ar--B, --(C═O) (CH₂)_(m) --B, and (b) A and B;wherein:Y--B is selected from the group consisting of ##STR104## A isselected from the group consisting of --(C═O)R¹¹, sialic acid, Kemp'sacid, quinic acid, --B, --SO₃ M, --OSO₃ M, --SO₂ NH₂, --PO₃ M'₂, --OPO₃M'₂, --NO₂, saturated or unsaturated carboxylic acids of 1 to 4 carbonatoms, optionally substituted with 1 to 2 hydroxyl groups, and esters,and amides of the carboxylic acid substitutents; W is selected from thegroup consisting of a covalent bond, --O--, --N, --S--, --NH--, and--NAc--; B is ##STR105## wherein U is selected from the group consistingof -R⁹, --CH₂ OR¹⁰, --CH₂ O-protecting group, --COOR¹¹, --CON(R¹¹)₂, and--COOM; R⁹ is lower alkyl; each n is independently selected from thegroup 0, 1, 2, and 3; each m is independently selected from the group 0,1, 2, 3, and 4; each q is independently selected from the group 0, 1,and 2; each s is independently selected from the group 1, 2, and 3; eachz is independently selected from the group 1 and 2; each t isindependently selected from the group 1 and 2, with the proviso thatwhen W is --N<, then t is 2, and for all other definitions of W, t is 1;R¹⁰ is selected from the group consisting of --H, --R¹¹, --SO₃ M,--(C═O)R¹¹, --SO₂ NH₂, --PO₃ M'₂, -alk--COOR¹³, alk--CON(R¹¹)₂ and--O-carbohydrate; R¹¹ is independently selected from the groupconsisting of --H, lower alkyl, cyclic alkyl of 5 to 6 carbon atoms,heterocyclic alkyl of 4 to 5 carbon atoms and 1 to 2 heteroatoms, loweraryl and lower aralkyl; R¹² is selected from the group consisting of--N(R¹¹)₂, and --SR¹¹ ; R¹³ is selected from the group consisting ofR¹¹, and M; R¹⁴ is selected from the group consisting of --H, and--OR¹⁰, with the proviso that when z is 2, then the two R¹⁴ groups takentogether with the carbon atoms to which each R¹⁴ group is attached mayform a double bond; R¹⁵ is independently selected from the groupconsisting of --R¹¹ and --COOH; M is selected from the group consistingof Na⁺, K⁺, Mg²⁺, and Ca²⁺ ; M' is selected from the group consisting of--H, --M, and R⁹ ; and X is selected from the group consisting of --O--,--S--, --C(R¹¹)₂ --, and --N(R¹¹)--; and pharmaceutically acceptablesalts thereof with the provisos that:(a) when any of R¹, R², R³, R⁴, R⁵,R⁶, R⁷, and R⁸ are --Y--B and W is a covalent bond, then at least oneadjacent position must be --OH or an ether moiety; (b) no more than twoof R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ may be Y--B when W is a covalentbond; (c) no more than three of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ maybe a covalent bond to A; (d) only one of R¹, R⁴, R⁵, and R⁸ may be acovalent bond to B; (e) when one of R¹, R⁴, R⁵, or R⁸ is a covalent bondto B, then no more than two other naphthyl substituents can be acovalent bond to A when A is not B; (f) at most three of R¹, R², R³, R⁴,R⁵, R⁶, R⁷, and R⁸ may be independently selected from the groupconsisting of --OH and ether moieties; (g) at least one of R¹, R², R³,R⁴, R⁵, R⁶, R⁷, and R⁸ is --H; (h) when any of R¹, R⁴, R⁵, and R⁸ is acovalent bond to B, then the adjacent position must be --OH or an ethermoiety; (i) at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ is asubstituent containing a B group, and at least one is a substituentcontaining an A group where A is not B; and (j) only when A is covalentbound to the naphthyl structure may A be --(C═O)R¹¹, and when A is--(C═O)R¹¹, at least one adjacent position must be --OH.
 2. Thecompounds of claim 1 wherein at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷,and R⁸ are independently selected from the group consisting of##STR106## --W(CH₂ --CR₁₁ (OR¹¹)CH₂ --B)_(t), --OH, lower alkoxy, loweraryloxy, lower aryloxy, lower alkoxyaryl, A and B; wherein W is acovalent bond or --O--, and t is
 1. 3. The compounds of claim 2 whereinat least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ are selected from thegroup consisting of ##STR107## --W(CH₂ --CR₁₁ (OR¹¹)CH₂ --B)_(t), --OH,and A where A is not B.
 4. The compounds of claim 1 wherein A and B areeach selected from the group consisting of sialic acid and fucose. 5.The compounds of claim 1 wherein A is selected from the group consistingof Kemp's acid, quinic acid, R and S forms of mandelic acid, R and Sforms of glyceric acid, R and S forms of lactic acid, propionic andacetic acid, and esters and amides any of the preceding acids --SO₃, and--PO₃ : ##STR108##
 6. The compounds of claim 5 wherein B is selectedfrom the group consisting of structure VI: ##STR109## wherein Me is amethyl group, R¹⁷, R¹⁸ and R¹⁹ are each independently --OH, --F,--N(R⁹)₂, wherein R⁹ is lower alkyl; inositol; substituted inositol;imidazole; substituted imidazole; benzimidazole; substitutedbenzimidazole; guanidine; pentaerythritol; substituted pentaerythritol;and substituted butane of the formula --CH₂ --CHR¹⁷ --CHR¹⁸ CH₂ R¹⁹wherein R¹⁷, R¹⁸ and R¹⁹ are independently OH, F or --N(R⁹)₂.
 7. Thecompounds of claim 1 wherein s is 1 or 2, R¹⁴ is --H or --OH, X is--O--, U is --CH₂ OR¹⁰ or -R⁹, and R¹⁰ is -alk--COOH, --SO₃ M, --H, or-alk--COOM.
 8. The compounds of claim 7 wherein s is
 2. 9. The compoundsof claim 8 wherein B is selected from the group consisting of fucose,galactose, mannose, and arabinose.
 10. The compounds of claim 1 whereins numbers are 1 and
 2. 11. The compounds of claim 10 wherein s is
 2. 12.The compounds of claim 1 wherein q numbers are 0 and
 1. 13. Thecompounds of claim 1 wherein m numbers are 0 and
 1. 14. The compounds ofclaim 1 wherein n numbers are 0 and
 3. 15. The compounds of claim 1wherein R¹⁰ is selected from the group consisting of --H, SO₃ M,-alk--COOR¹³, and --O--carbohydrate.
 16. The compounds of claim 15wherein R¹⁰ is selected from the group consisting of --H, --SO₃ M, and-alk--COOR¹³.
 17. The compounds of claim 1 wherein R¹¹ is selected fromthe group consisting of --H, lower alkyl, and lower aralkyl.
 18. Thecompounds of claim 17 wherein R¹¹ is --H.
 19. The compounds of claim 1wherein R¹² is --N(R¹¹)₂.
 20. The compounds of claim 1 wherein R¹⁴ isselected from the group consisting of --H and --OH.
 21. The compounds ofclaim 1 wherein R¹⁵ is selected from the group consisting of --COOH,--H, and --CH₃.
 22. The compounds of claim 1 wherein M is Na⁺.
 23. Thecompounds of claim 1 wherein M' is selected from the group consisting of--H, Na⁺, and --CH₃.
 24. The compounds of claim 1 wherein X is --O--.25. The compounds of claim 1 wherein U is selected from the groupconsisting of --CH₂ OR¹⁰ and -R⁹.
 26. The compounds of claim 1 wherein Wis selected from the group consisting of a covalent bond and --O--, andt is
 1. 27. The compounds of claim 3 wherein at least one of R¹, R², R³,R⁴, R⁵, R⁶, R⁷, and R⁸ are independently selected from the groupconsisting of --W(CH₂ (C═C(R¹⁵)₂)--CH₂ --B_(t) where W is a covalentbond or --OH, t is 1, and R¹⁵ is independently --H, --CH₃, and COOH. 28.The compounds of claim 1 wherein R³ and R⁶ are selected from the groupconsisting of --SO₃ M, --OSO₃ M, --NO₂, and --CO₂ H, and at least one ofR¹, R², or R⁷ is selected from the group OH and --NH₂.
 29. The compoundsof claim 1 wherein R¹ is a covalent bond to B, R² and R³ are selectedfrom the group consisting of --OH, ##STR110## where W is a covalent bondand t is 1, R⁷ is selected from the group consisting of --H or --OH, andR⁶ is selected from the group consisting of --H and --(CH₂)_(m)--(CHR⁹)_(q) --(CH₂)_(m) --A, where each m and q are independentlyselected from 0 or 1, and A is a saturated or unsaturated carboxylicacid of 1 to 4 carbon atoms, optionally substituted with 1 to 2 hydroxygroups, and esters and amides thereof.
 30. The compounds of claim 29wherein s is 1 or 2, R¹⁴ is --H or --OH, X is --O--, U is --CH₂ OR¹⁰ or-R⁹, and R¹⁰ is -alk--COOH, --SO₃ M, --H, or -alk--COOM.
 31. Thecompounds of claim 29 wherein R¹⁵ is --H.
 32. A method of treating aselectin-mediated disorder selected from the group consisting of cancer,auto-immune disorders, and inflammation, comprising the stepof:administering to a patient in need thereof a therapeuticallyeffective amount of a compound of claim
 1. 33. A pharmaceuticalcomposition comprising at least one compound of claim 1 and apharmaceutically acceptable carrier, wherein at least one compound ofclaim 1 is bound to a pharmaceutically active drug.